After a radiological incident, there is an urgent need for fast and reliable bioassays to identify radiation-exposed individuals within the first week post exposure. This study aimed to identify candidate radiation-responsive protein biomarkers in human lymphocytes in vivo using humanized NOD scid gamma (Hu-NSG) mouse model. Three days after X-irradiation (0–2 Gy, 88 cGy/min), human CD45+ lymphocytes were collected from the Hu-NSG mouse spleen and quantitative changes in the proteome of the human lymphocytes were analysed by mass spectrometry. Forty-six proteins were differentially expressed in response to radiation exposure. FDXR, BAX, DDB2 and ACTN1 proteins were shown to have dose-dependent response with a fold change greater than 2. When these proteins were used to estimate radiation dose by linear regression, the combination of FDXR, ACTN1 and DDB2 showed the lowest mean absolute errors (≤0.13 Gy) and highest coefficients of determination (R2 = 0.96). Biomarker validation studies were performed in human lymphocytes 3 days after irradiation in vivo and in vitro. In conclusion, this is the first study to identify radiation-induced human protein signatures in vivo using the humanized mouse model and develop a protein panel which could be used for the rapid assessment of absorbed dose 3 days after radiation exposure.

Following a large-scale radiological incident, there is a need for FDA-approved biodosimetry devices and biomarkers with the ability to rapidly determine past radiation exposure with sufficient accuracy for early population triage and medical management. Towards this goal, we have developed FAST-DOSE (Fluorescent Automated Screening Tool for Dosimetry), an immunofluorescent, biomarkerbased system designed to reconstruct absorbed radiation dose in peripheral blood samples collected from potentially exposed individuals. The objective of this study was to examine the performance of the FAST-DOSE assay system to quantify intracellular protein changes in blood leukocytes for early biodosimetry triage from humanized NOD-scid-gamma (Hu-NSG) mice and non-human primates (NHPs) exposed to ionizing radiation up to 8 days after radiation exposure. In the Hu-NSG mice studies, the FAST-DOSE biomarker panel was able to generate delivered dose estimates at days 1, 2 and 3 post exposure, whereas in the NHP studies, the biomarker panel was able to successfully classify samples by dose categories below or above 2 Gy up to 8 days after total body exposure. These results suggest that the FAST-DOSE bioassay has large potential as a useful diagnostic tool for rapid and reliable screening of potentially exposed individuals to aid early triage decisions within the first week post-exposure. In the event of a large-scale radiological incident or accident, hundreds of thousands of people may be exposed to ionizing radiation. There is an important need for the development of FDA-approved point-of-care radiation biodosimeters and in vitro diagnostic devices (IVDs) with the ability to rapidly determine past radiation exposure with sufficient accuracy for early population triage and medical management. Biodosimetry technologies may be designed for early in-the-field triage (usually qualitative) or for more clinical evaluation and medical management that includes dose level confirmation (usually quantitative or semi-quantitative) 1. We have recently developed a new high-throughput biodosimetry assay system called FAST-DOSE (Fluorescent Automated Screening Tool for Dosimetry) which is designed for rapid immune-detection and quantitation of radiation responsive protein biomarkers and reconstruction of dose in human peripheral blood leukocytes. This biomarker-based triage assay has large potential as a useful diagnostic tool for the mass-screening of potentially exposed individuals and offers a short "time-to-result" since cell culture is not required compared with the gold standard micronucleus and dicentric biodosimetry assays 2-4. The FAST-DOSE assay device is based on imaging flow cytometry (IFC) 5-7 and a panel of intracellular biomarkers identified by earlier proteomic study 8 to rapidly quantify the upregulation of biomarker expression in blood leukocytes using fluorescent imagery and algorithms for the estimation of absorbed dose. The advantage of IFC (ImageStream, Luminex, Austin, TX) technology is that it combines the speed and quantifica...

The mouse (Mus musculus) is an extensively used model of human disease and responses to stresses such as ionizing radiation. As part of our work developing gene expression biomarkers of radiation exposure, dose, and injury, we have found many genes are either up-regulated (e.g. CDKN1A, MDM2, BBC3, and CCNG1) or down-regulated (e.g. TCF4 and MYC) in both species after irradiation at ~4 and 8 Gy. However, we have also found genes that are consistently up-regulated in humans and down-regulated in mice (e.g. DDB2, PCNA, GADD45A, SESN1, RRM2B, KCNN4, IFI30, and PTPRO). Here we test a hematopoietically humanized mouse as a potential in vivo model for biodosimetry studies, measuring the response of these 14 genes one day after irradiation at 2 and 4 Gy, and comparing it with that of human blood irradiated ex vivo, and blood from whole body irradiated mice. We found that human blood cells in the hematopoietically humanized mouse in vivo environment recapitulated the gene expression pattern expected from human cells, not the pattern seen from in vivo irradiated normal mice. The results of this study support the use of hematopoietically humanized mice as an in vivo model for screening of radiation response genes relevant to humans.

Heightened threats for nuclear terrorism using improvised nuclear devices (IND) necessitate the development of biodosimetry assays that could rapidly assess thousands of individuals. However, the radiation exposures from an IND may be complex due to mixed fields of neutrons and photons (γ-rays), shielding from buildings, and proximity to the epicenter among others. In this study we utilized lipidomics to analyze serum samples from mice exposed to various percentages of neutrons and X-rays to a total dose of 3 Gy. Triacylglycerides, phosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylcholines (LPCs), sphingolipids, and cholesteryl esters all showed delayed increases at day 7 compared to day 1 after irradiation, while diacylglycerides decreased in mixed field exposures and phosphatidylcholines (PCs) remained largely unchanged. Individual lipid molecules with a high degree of unsaturation exhibited the highest fold changes in mixed fields compared to photons alone. More importantly, the increased ratio of LPCs to PCs of each irradiation group compared to control could be used as a radiation biomarker and highlights the existence of a pro-inflammatory phenotype. The results showed that even a small percentage of neutrons in a mixed field can lead to high biological responses with implications for accurate biodosimetry, triage and medical managements of exposed populations.

Biodosimetry-based individualized reconstruction of complex irradiation scenarios (partial-body shielding and/or neutron + photon mixtures) can improve treatment decisions after mass-casualty radiation-related incidents. We used a high-throughput micronucleus assay with automated scanning and imaging software on ex-vivo irradiated human lymphocytes to: a) reconstruct partial-body and/or neutron exposure, and b) estimate separately the photon and neutron doses in a mixed exposure. The mechanistic background is that, compared with total-body photon irradiations, neutrons produce more heavily-damaged lymphocytes with multiple micronuclei/binucleated cell, whereas partial-body exposures produce fewer such lymphocytes. To utilize these differences for biodosimetry, we developed metrics that describe micronuclei distributions in binucleated cells and serve as predictors in machine learning or parametric analyses of the following scenarios: (A) Homogeneous gamma-irradiation, mimicking total-body exposures, vs. mixtures of irradiated blood with unirradiated blood, mimicking partial-body exposures. (B) X rays vs. various neutron + photon mixtures. The results showed high accuracies of scenario and dose reconstructions. Specifically, receiver operating characteristic curve areas (AUC) for sample classification by exposure type reached 0.931 and 0.916 in scenarios A and B, respectively. R2 for actual vs. reconstructed doses in these scenarios reached 0.87 and 0.77, respectively. These encouraging findings demonstrate a proof-of-principle for the proposed approach of high-throughput reconstruction of clinically-relevant complex radiation exposure scenarios.