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 Table of Contents  
REVIEW ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 4  |  Page : 132-146

Acute radiation syndrome: An update on biomarkers for radiation injury


1 Department of Pharmacology and Molecular Therapeutics, Division of Radioprotectants, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences; Department of Scientific Research, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, USA
2 Tech Micro Services, 4417 Maple Avenue, Bethesda, MD, USA

Date of Web Publication12-Mar-2019

Correspondence Address:
Vijay K Singh
Department of Pharmacology and Molecular Therapeutics, Division of Radioprotectants, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences; Department of Scientific Research, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_26_18

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  Abstract 


The possible detonation of a radiological dispersal device or improvised nuclear device in a metropolitan city, or the accidental exposures to a radiation source, nuclear accidents, or the all-to-often threats of radiological/nuclear terrorism have led to the urgent need to develop essential analytic tools to assess such radiation exposures, especially radiation doses to exposed individuals. This exposure-assessing work using biological samples, and discipline, is known as biodosimetry. As of late, this field has progressed significantly as it has made use of the advances within newer areas of biologic analytics, namely omics (genomics, proteomics, metabolomics, and transcriptomics), lymphocyte kinetics, optically stimulated luminescence, and electron paramagnetic resonance technology in addition to conventional cytogenetic techniques. The use of automated high throughput platforms and the planning for laboratory surge capacity during the time of need are the latest developments in the field of biomarkers for biodosimetry. Such biomarkers are also needed for radiation exposure/dose conversion estimates that are essential for the development and application of radiation countermeasures, from animals to humans and that are currently being developed following the US Food and Drug Administration Animal Rule. Here, we present and discuss the current status of various biomarkers for assessing radiation dose after radiation exposure. It is anticipated that with the advent of improved biomarkers and associated biomarker platforms for the acute radiation syndrome, exposed victims can be more efficiently triaged and appropriately treated than is currently allowable. The latest advances in the field, and identify the areas where improvement is needed are also listed and discussed.

Keywords: Acute radiation syndrome, animal rule, biomarkers, chromosomal aberration, irradiation, mice, nonhuman primates, omics


How to cite this article:
Singh VK, Santiago PT, Simas M, Garcia M, Fatanmi OO, Wise SY, Seed TM. Acute radiation syndrome: An update on biomarkers for radiation injury. J Radiat Cancer Res 2018;9:132-46

How to cite this URL:
Singh VK, Santiago PT, Simas M, Garcia M, Fatanmi OO, Wise SY, Seed TM. Acute radiation syndrome: An update on biomarkers for radiation injury. J Radiat Cancer Res [serial online] 2018 [cited 2019 May 19];9:132-46. Available from: http://www.journalrcr.org/text.asp?2018/9/4/132/253999




  Introduction Top


A biomarker is an objective feature that can be precisely assessed, and that can specify a specific biological, pathological, or therapeutic development of the host. These features include, but are not limited to the following: specific genomic sequences, γ-H2AX (phosphorylated form of a variant of the H2A protein family), messenger RNA (mRNA), cell surface receptor expression patterns, complete blood count (CBC), differential for lymphocyte depletion kinetics, microRNA (miRNA), long noncoding RNAs (LncRNAs), proteins, cytokines and growth factors, metabolites/lipidomes, microbiota, premature chromosome condensation (PCC), dicentrics, electron paramagnetic resonance (EPR) or optically stimulated luminescence of teeth, radiographic or other imaging-based measurements, and electrocardiographic parameters [Figure 1].[1],[2] Biomarkers are needed to assess the absorbed dose of radiation after a radiological or nuclear accident or deliberate radiation exposure event.[3],[4],[5],[6],[7],[8] The nuclear reactor accidents of Chernobyl and Fukushima-Daiichi are reminders that human error or natural disasters can lead to an accident with lasting effects on both health and the environment. Improper disposal of old radiological equipment used in the diagnosis of human diseases or laboratory work and improper use of radioactive materials can lead to exposure of individuals and contamination.[9] The bombings of Hiroshima and Nagasaki demonstrated the catastrophic effects of nuclear weapons used in times of war.
Figure 1: Various approaches for biomarker identification and validation for radiation injury. Different technologies have been used for identifying and validating biomarkers for radiation injury. Samples from in vitro studies, various animal models, and humans have been used

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There are a large number of recent studies using various strategies to identify and validate the biomarkers for both total-body and partial-body absorbed doses of radiation.[1],[2],[4] Ionizing radiation (IR) can cause injury to cells and tissues, and if such irradiation is intense and the injury sustained is severe, multi-organ involvement may develop, with subsequent multi-organ failure and ultimately death.[2] In general, the sensitivity of given cell types and tissues to IR is directly proportional to mitotic rates and rates of cell/tissue “turnover” within the body and conversely, indirectly proportional to the extent of cell/tissue differentiation, i.e., rapidly dividing cells within tissues having high turnover rates are generally highly radiosensitive, whereas nondividing or very slowly dividing cells of tissues that turn over more slowly and are more differentiated tissues are relatively resistant and less sensitive to acute IR. Cells of the hematopoietic system, gametocytes within reproductive tissues, and the lining cells of the intestine are the most sensitive to IR while the cells of the central nervous system are significantly resistant. It is highly unlikely that generic biomarkers for all types of radiation-induced injuries, under all types of possible exposure scenarios, will be identified and developed for diagnostic purposes in the near future. Simply stated, there is no perfect biomarker at this time to judge the absorbed radiation dose and assess the extent of injury of the host. It is important that complementary and surrogate methods of biodosimetry must be developed and implemented for filling this critical gap in our knowledge.

In a mass casualty scenario, it will be a challenging task for medical professionals and first responders to triage radiation-exposed victims into definable and treatable groups. Under such situation, individuals exposed to minimal IR doses (<1 Gy) may not need immediate medical attention. However, individuals exposed to elevated, but still considered moderate (relative to survivability) doses (1–6 Gy) and that lead to either the hematopoietic subsyndrome or to the gastrointestinal (GI) subsyndrome at a higher range of doses (6–10 Gy) will benefit (in terms of increased survivability) from the timely administration of treatments and the use of other supportive care measures currently considered as “standard of care.”[10],[11] At present, those exposed individuals with estimated doses in the lethal range (>10 Gy) and that fall subject to the neurovascular subsyndrome, unfortunately, cannot be effectively treated to promote survival, but would receive palliative care to improve “quality of life.” The capacity to identify these clinical subcategories among acutely IR exposed individuals is critical for the conservation of scarce medical resources during any mass casualty scenario. Current strategies to assess the status of IR exposed individuals are based on both physical dosimetric information (if and when possible) and biodosimetry (cytogenetics), or, most importantly, on the clinical signs and symptoms of the exposed individual over time. Such signs and symptoms include estimates of (a) the time of onset and severity of nausea and vomiting, (b) cytogenetic analysis of the dicentrics, chromosomal aberration, (c) micronuclei assay, and (d) kinetics of leukocyte depletion after radiation exposure. Cytogenetic analysis for dicentrics and lymphocyte kinetic assays are both labor-intensive, time-consuming, and difficult to execute for a large number of victims in a mass casualty scenario, especially over a short period and often with constrained resources. Optimally, with a biodosimetry device (still under development), results should be available in <15 min and cover a dose range of 0.5–10 Gy.[12] Thus, there is an urgent need to develop and validate noninvasive or minimally invasive biomarkers for radiation exposure based on molecular changes such as DNA, RNA, protein, and metabolites, which may be less time consuming, labor intensive, and adaptable as a high throughput technique.[2],[3] Currently, several biomarkers are approved for various injuries by the U. S. Food and Drug Administration (US FDA), the European Medicines Agency, and the Japanese Pharmaceuticals and Medical Devices Agency, but none of the approved biomarkers by these regulatory agencies are for the radiation-induced injuries. Multiple potential biomarkers for radiation injury are being identified and validated by a large number of investigators in various laboratories in different countries.[13],[14],[15]

In addition to the need to further identify and to develop biomarkers of radiation injury during a mass casualty scenario, there is also a need for specific biomarkers for radiation countermeasure development, specifically for countermeasures for acute radiation syndrome. Such biomarkers are used as a trigger for intervention, in deciding the optimal drug dose for humans based on experiments conducted in animal models, and for decisions related to the optimal treatment regimen for a given drug. The human dose of the countermeasure needs to be decided based on biomarkers since such agents, for ultimate regulatory approval, must follow the path of the FDA Animal Rule where final efficacy testing is conducted in well-controlled studies using animal models. Further, biomarkers are also helpful in understanding the mechanism of action of radiation countermeasures. In this article, we discuss only biomarkers for radiation injuries. In this regard, we list and discuss various classes of biomarkers identified in different models (mice, rats, minipigs, nonhuman primates (NHPs), and humans) and various biosamples (urine, blood, saliva, fecal material, and sebum) used for identifying and validating biomarkers. The following areas have been extensively studied in various laboratories for identifying biomarkers.


  Peripheral Complete Blood Count and Lymphocyte Kinetics Top


CBCs can be a strong indicator of absorbed radiation dose when blood samples collected either at a single time point or serially at different time points post-irradiation. The peripheral blood cells analyzed are lymphocytes, leukocytes, granulocytes/neutrophils, and platelets.[2],[16],[17] The correlation between CBCs and absorbed radiation dose (acute radiation doses) exists not only in the early time window (1 or 2 days) but also during the delayed phase (up to 4 weeks) after radiation exposure.[18] For an optimal assessment of absorbed radiation dose, CBC should be obtained soon after radiation exposure. CBC has been used to monitor the health of the radiation-exposed victims of many accidents as a diagnostic tool.[9],[19],[20] In addition to assessing acute radiation injury, CBC assays can be used for analyzing several other clinical indications. Although such assays are quite useful, easy to establish, readily automated, relatively inexpensive and available, they are limited in terms of assessing radiation exposures that are acute (not chronic or intermittent) by nature and most commonly are sparsely ionizing and deeply penetrating radiation and that affect large bodily areas or bodily volumes. It is well known that increasing the intensity and dose of the IR exposure, increases the extent and severity of neutropenia and thrombocytopenia in various animal models and humans.[17],[21],[22] Thrombocytopenia is considered a better predictor of mortality than neutropenia.[2]

The rates of lymphocyte depletion following acute IR exposure appear to be a useful hematological parameter for determining the radiation dose under select conditions of radiation exposure. The Biodosimetry Assessment Tool incorporates clinical indications with lymphocyte depletion kinetics to predict the dose.[23] The depletion of lymphocytes, an abortive rise in neutrophil count, and an increased ratio of neutrophils to lymphocytes can provide valuable information for exposure to high radiation doses.[24]


  Proteomics Top


Proteomics is a growing field of biological research, and proteomics-based approaches are promising to be deployed as point-of-care diagnostic tools, and such effort can be translated to high-throughput system for biodosimetry. Several studies have demonstrated the correlation between the expression of various proteins and the dose of acute radiation exposure.[1],[25] Over the last two decades, changes in cytokines, chemokines, and other protein molecules have been extensively investigated.[1],[26],[27]

Cytokines, chemokines, growth factors

Several cytokines, chemokines, and growth factors have been identified as candidate protein biomarkers of radiation injury over the last several years using total-and partial-body acute radiation exposure in the murine and NHP models.[28],[29],[30],[31] Interleukin-6 (IL-6) and growth factors such as granulocyte colony-stimulating factor (G-CSF) have been shown to be up-regulated in irradiated mice and NHPs and play an important role in mediating radiation injury.[32],[33],[34],[35],[36],[37],[38],[39] This is a significant and active area of research, with several laboratories working on attempting to validate several cytokines/growth factors as biomarkers for radiation injuries and absorbed radiation dose.

IL-18 has been shown to be up-regulated in several organs collected from CD2F1 mice exposed to 5–12 Gy 60Co γ-radiation. IL-18 was found to be elevated in mouse thymus, spleen, and BM cells after total-body irradiation. Furthermore, IL-18 serum concentrations showed a direct correlation to acute radiation dose in minipigs, NHPs, and mice.[40] In addition to IL-18, IL-18 receptor-α (IL-18Rα) and IL-18 binding protein (IL-18BP, endogenous antagonist of IL-18) have been shown to change in response to radiation exposure using the murine model. IL-18BP increased on day 1 after irradiation in mice and returned to baseline within 3 days after 5–7 Gy exposure, and within 7 days after 8 Gy. High doses of radiation, 9 or 10 Gy, sustained a higher level of IL-18BP in mouse serum.[41] In a recent study, increased levels of IL-18 in NHP urine has been reported.[42] There is also a report demonstrating elevated levels of several cytokines (IL-5, IL-10, IL-12, and IL-18) in B6D2F1 mice exposed to a mixed field of neutrons and γ-rays.[43] Levels of a few cytokines (IL-18), chemokines, and growth factors (G-CSF) in animals after irradiation are correlated with the dose of radiation exposure, but limited changes in dose rate have no effect on such factors.[44]

There are several studies for cytokines using samples from patients undergoing radiotherapy for malignancies. A dose-response relationship was reported for the expression of IL-4, IL-6, IL-8, epidermal growth factor, vascular endothelial growth factor, monocyte chemoattractant protein-1, and tumor necrosis factor-α in saliva from patients undergoing head and neck radiotherapy.[45],[46] There is also a report about the correlation between insulin growth factor-1 (IGF-1) levels and resistance to irradiation in the minipig model.[47] We investigated the levels of IGF-1 in serum samples obtained from NHPs exposed to 60Co γ-radiation. IGF-1 levels were compared between survivors and decedents. There was no significant differences between the IGF-1 levels of the two groups at any of the compared time points [Figure 2].
Figure 2: Comparison of NHPs insulin growth factor-1 levels in serum samples of survivors and decedents after exposure to radiation. Animals were exposed to 5.8, 7.2, or 7.6 Gy (0.6 Gy/min, 60Co γ-radiation), and blood samples for serum were collected at various time points in relation to irradiation and were quantified using ELISA. The data for each time point are shown as the mean ± standard error (n = 18 for 7.2 Gy; n = 31 for 5.8 Gy; n = 10 for 7.2 Gy; n = 10 for 7.6 Gy)

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γ-H2AX

H2AX is a variant of the H2A protein family, a component of the histone octamer in nucleosomes and it is phosphorylated by kinases, for example, ataxia telangiectasia mutated (ATM) and ATM-Rad3-related PI3K pathway.[48] When DNA damage leads to double-strand breaks, it is followed by histone H2AX phosphorylation at Serine 139. This phosphorylated H2AX is known as γ-H2AX, and this is the initial step in recruiting and localizing DNA repair proteins. Regions in chromatin with γ-H2AX are conveniently detected by immunofluorescence microscopy or flow cytometry and serve as signals of DSBs. This is an efficient method for estimating the radiation dose for partial- and total-body irradiation. The assay has been used in small animal models, NHPs, and humans.[49],[50],[51],[52]

Regions in chromatin with γ-H2AX are conveniently detected by immunofluorescence and flow cytometry microscopy and serve as signals of DSBs.

Other proteins

Among the other proteins that might serve as potential biomarkers for radiation exposure are C-reactive protein (CRP), serum amyloid A (SAA), growth arrest and DNA damage-inducible 45 (GADD45) proteins, FMS-like tyrosine kinase 3 ligands (flt3 L), and salivary α–amylase.[28],[30] CRP and SAA were the first identified protein biomarkers of radiation exposure. Elevated levels of SAA has been demonstrated in patients undergoing total- or partial-body radiotherapy and in victims of Tokai-Mura criticality accidents.[53],[54],[55] Based on reports in the literature, there appear to be good correlations between the elevation of CRP, increasing doses of radiation, and the initiation of acute radiation syndrome.[56],[57]

Recently, there are a large number of studies using various techniques such as Western blot, MALDI-MS, 2D-DIGE analysis to identify proteins responsive to radiation exposure.[45],[46],[58],[59],[60]


  Cytogenetics Top


Dicentrics, micronuclei, and premature chromosome condensation

Cytogenetic techniques currently being explored for biodosimetric purposes include the estimation of chromosomal aberrations or dicentric assay, the PCC assay, the micronucleus assay or cytokinesis-block micronucleus assay, and fluorescence in situ hybridization assay. IR exposure of sufficient intensity can lead to both single and double strand DNA breaks. A dicentric chromosome (an unstable chromosome with 2 centromeres) may be formed due to an error or abnormal chromosome replication during the repair process after radiation exposure.[61] The dicentric cytogenetic assay is well established and is considered by the radiation biology research community to be the gold standard for cytogenetic-based biodosimetry following acute irradiation. This assay has been endorsed as an indicator of radiation injury by the International Atomic Energy Agency in the event of a radiation emergency. Since it requires highly trained technical staff and sophisticated equipment that may not be available at the time of need, this assay may not be the optimal choice to assess exposure doses of individuals in a mass casualty situation. To get an accurate dose of exposure from a metaphase spread, a dose-response calibration curve needs to be ascertained.

Another well-known cytogenetic technique for dosimetry is the micronucleus assay. Micronuclei serve as an important parameter for measuring chromosomal damage caused by IR. Micronuclei are fragments or whole centric chromosomes or chromatids and are generated by nonrepaired or misrepaired DSBs in anaphase. The frequency of micronuclei detected in peripheral blood lymphocytes is directly linked to damage caused by irradiation and a predictor of the degree of damage due to radiation exposure. It is regarded as the most appropriate cytogenetic assay to assess the radiation exposure dose in the case of a mass-casualty scenario.[62] The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is a well standardized and validated technique.

The PCC assay can be executed soon after irradiation and data can be generated within 3–4 h as compared to the other, more slowly developing cytogenetic assays. It appears to be the most suitable assay for estimating doses of exposure soon after blood sampling. It is possible to accurately investigate the effect of low and high doses of acute exposure to a variety of radiation qualities (e.g., to either low- or high-linear energy transfer [LET] IR). Furthermore, it has been documented experimentally that PCC also has the potential to discriminate accurately between total- and partial-body exposures.[63],[64]

The fluorescence in situ hybridization chromosome painting has been used to measure the frequency of stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers.[65] The frequency of such “stable chromosomal translocations” is thought to reflect the degree of radiation genetic injury; in turn, the relative injurious radiation dose received.


  Citrulline Top


Radiation injury to the gut remains an important clinical issue for which hardly any therapeutic strategy exists. Citrulline is linked to radiation injury of the gut. It is a nitrogen end product of glutamine metabolism in small-bowel enterocytes. It has been identified as a potential circulating biomarker for radiation-induced GI injury and epithelial cell loss as a result of radiation exposure. In brief, citrulline levels in circulation reflect the overall integrity of-and the extent of injury to the gut enterocyte population. Citrulline levels are tissue-specific for small-bowel epithelium, and its plasma concentration has been inversely related to GI tissue damage. The decreased intestinal absorptive function following irradiation of the host is due to the loss of gut enterocytes which constitutes the absorptive mucosal surface. The correlation between radiation exposure-induced epithelial cell loss and plasma citrulline level has been well documented in various animal models, and several investigators are currently working to validate this biomarker for radiation injury.[66],[67],[68] In NHPs exposed to potentially lethal doses of γ-radiation (5.8 and 6.5 Gy), reduction in citrulline levels was not observed: This contrasted the observations made at significantly higher radiation doses that suppressed citrulline levels. In the former case (of the lower IR doses), enterocyte damage may not have been substantial enough to lower citrulline level in the peripheral circulation. However, exposure to 7.2 Gy total-body γ-radiation reduced plasma citrulline levels in NHPs. There are several reports demonstrating reduced levels of citrulline in irradiated mice, minipigs, and NHPs.[40],[69],[70],[71],[72]


  Tooth Enamel and Fingernail Based Biomarkers Top


Electron magnetic resonance (EPR) or electron spin resonance spectroscopy (ESR) represents an alternative method, a physiochemical analytic method, for estimating radiation dose. The EPR-based analytic method employs magnetic resonance spectroscopy to determine radiation-induced free radical formation in tooth enamel, bone, and nails. EPR-based radiation dosimetry techniques are non or minimally invasive and free of biological confounders (disease and trauma) often associated with other biological samples. Radiation exposure results in the generation of free radicals inducing oxidative stress and triggering damage to proteins, lipids, and DNA. Usually, the life span of the unpaired electrons is of nanoseconds in biological tissues. Such radiation-induced changes can be fixed in calcified tissues and can be detected by EPR at a later time. Tooth enamel contains 97% hydroxyapatite, and the radiation-induced free radicals are captured in the hydroxyapatite lattice after radiation exposure. Furthermore, radiation-induced free radicals can be fixed into the α-keratin of fingernails and hair.[73] The EPR spectroscopy is well established as a viable technique for measuring free-radicals in biological samples. Recent developments with EPR techniques offer potential opportunities for clinical dosimetry that could be deployed for triage in a mass causality scenario.[74],[75] The EPR can also be used to assess partial-body exposure by measurement of tooth and nail samples from different limbs. This technique has recently been used in accident victims with exposure to the hand.[76],[77] The EPR is efficient technology with a read time of approximately 5 min per sample.

ESR has been used to investigate the potential of human hair samples for use in biological dosimetry. The hair samples were irradiated at low doses (5–50 Gy) and high doses (75–750 Gy) from a γ-radiation source at a dose rate of 0.25 Gy/s.[78] The linear dose-response curves at low doses saturated after ~300 Gy. The EPR signal intensities of samples fell to their half-values in 44 h in black hair, 41 h in blonde and brown hairs, 35 h in dyed black hair, and 17 h in red hair. In brief, results of this study suggest that hair samples can be used as a biological dosimetry.


  Metabolomics and Lipidomics Top


The discipline of metabolomics consists of the study of the metabolome and the global changes in the metabolite products. It is a promising field and is known for the rapid, qualitative, and quantitative assessment of small molecules of <1 kDa in any biofluid or tissue. Lipidomics is the full assessment of changes in lipids and considered as a component of metabolomic analyses. Global approaches of metabolomics allow for the full scanning of the metabolomes and pattern identification based on pathway interactions. Targeted approaches are more quantitative and concentrate on specific metabolites or changes along a metabolic pathway. Easily accessible biofluids such as urine, blood, and saliva have been the primary focus of metabolomics biodosimetry, and such biofluids require minimal invasive techniques to obtain the samples after any radiological/nuclear event. In a recent article by Pannkuk et al. metabolomics changes after irradiation in various animal models using different radiation sources as well as in humans have been elegantly discussed.[3] Nuclear magnetic resonance (NMR) is the platform of choice since it provides data on the structure of a biomarker. The major weaknesses of mass spectrometry (MS) are the major strengths of NMR spectroscopy [Table 1].[79],[80],[81] A large number of metabolites have been identified and studied in different models using NMR platform.[82] Chen et al.[83] have identified a broad range of metabolic changes, including 2-oxoglutarate, taurine, N-methyl-nicotinamide, hippurate, choline, creatine, succinate, methylamine, and citrate. This study was conducted with noninvasive urine samples collected from mice exposed to 8.0 Gy whole-body radiation over a period of 7 days and also from unirradiated control mice. There are several databases for NMR-based metabolomic studies.[80] However, this technique appears to miss many metabolites within a given sample that could well serve as biomarkers of radiation injury. Liquid chromatography coupled to MS (LC-MS) provides high selectivity, chromatographic separation as well as identification of molecules with variable polarity. The high-performance LC coupled with time-of-flight MS (TOFMS) significantly increased chromatographic resolution and sensitivity to improve the ability to identify additional ions present in low abundance. Compared to the NMR which can assess 50–100 high abundance metabolites, LC-TOFMS can assess thousands. Furthermore, gas chromatography-MS is focused on identification of thermally stable metabolites. Though LC-TOFMS is complementary to LC-MS, the labor-intensive sample preparation severely limits its utility. The global metabolomic profiling appears complex but the targeted approaches used in radiation biodosimetry are more simple.[83],[84],[85] In one study, urine samples collected from patients exposed to whole-body radiation (a single dose of 1.25 Gy, ~0.1 Gy/min, 15 MV X-rays) were analyzed for their global metabolomic profile with ultra-performance LC coupled to TOFMS.[86] Seven markers, trimethyl-L-lysine, carnitine conjugates acetylcarnitine, decanoylcarnitine, octanoylcarnitine, hypoxanthine, xanthine, and uric acid showed differences between pre- and post-exposure samples. Furthermore, analysis demonstrated sex differences in the patterns of excretion of the markers. This was the first radiation metabolomics study with human urine samples.
Table 1: The advantages and limitations of mass spectrometry spectrometry and nuclear magnetic resonance spectroscopy as an analytical tool for metabolomics

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Over the last 10 years, extensive metabolomic studies have been conducted using various animal models such as mice (several strains), rats, minipigs, NHPs, and in humans. For this kind of metabolomic analysis, samples of urine, plasma, serum, saliva, feces, and sebum have been used. Recently, an extensive review article has been published summarizing all past studies with metabolomics.[3] A comparison between metabolomic data obtained from analysis of serum samples from NHPs following exposure to either 6.5 Gy or 7.2 Gy total-body 60Co γ-radiation, with samples collected at 24 h post-irradiation, revealed distinct separations based on multivariate data models and higher compound fold changes with 7.2 Gy.[87],[88],[89] In a very recent study using NHPs exposed to different doses of total-body 60Co γ-radiation, metabolites of plasma and exosomes were compared. Based on the differences in metabolite composition between plasma and exosomes (i.e., extruded, extracellular microvesicles), it appears that exosomal profiling would enable detection and identification of low abundance metabolites that comprise exosomal cargo that would otherwise get obscured with plasma profiling or would require a large quantity of plasma to be processed.[26] These results warrant further investigations to know the functional and signaling roles that exosomes may have in mediating the systemic effects of radiation exposure.

Citrulline is also a metabolite and reliable biomarker of small bowel enterocyte mass. Keeping in mind its importance as a biomarker of intestinal injury, it is discussed above separately.


  Genomics and Transcriptomics Top


Genomics and transcriptomics are two promising areas of research. Genomics is the discipline of investigating changes in global genome expression using recombinant DNA technology and bioinformatics. Transcriptomics is the investigation of the complete RNA products encoded by the genome. Both of these relatively new research areas are being currently investigated for potential applications in the field of biodosimetry. Several groups are working using different animal models and various types of RNAs.

Genome and messenger RNA

Microarray analysis using blood samples from total-body irradiated mice, clinical patients, and ex vivo irradiated human blood samples was performed with the intent of identifying specific gene expression signatures of radiation exposure. The gene expression profiles from the ex vivo irradiated human samples, and clinically irradiated cancer patient samples were correlated while gene expression from mice differed. This study also provided information to predict the radiation dose of the exposure in both ex vivo and clinical total-body irradiated samples.[90],[91] In addition to total-body-irradiation, studies with partial-body exposure are equally important since there is the probability for such exposure during a radiological/nuclear event. The gene expression profiles were studied in the blood samples collected from partial-body-irradiated mice. Mice were irradiated with 0.5, 2, and 10 Gy. Findings were able to distinguish radiation exposed mice from unexposed mice. The gene profiles from total-body irradiation were not predictive of partial-body irradiation.[92]

During the last few years, several gene expression studies have been conducted with samples obtained from irradiated (60Co source) baboons.[93],[94],[95],[96] These baboons received either partial-or total-body irradiation. Such gene expression studies have been used to correlate different levels of hematologic acute radiation syndrome (HARS) such as H0, H1, H2, and H3. In one study, 22 genes were confirmed H1-3 identification and seven genes for identification of H2-3 using qRT-PCR. For H1-3 classifications, genes were three to 5-fold down-regulated compared to H0 on the first 2 days after irradiation.[93] Some genes were 10.3-fold (VSIG4) or even 30.7-fold up-regulated (CD177) compared to H0. For H2-3, some genes appeared four to 7-fold up-regulated relative to H0 (RNASE3, DAGLA, ARG2). Other genes showed 14–33-fold down-regulation over H0 (WNT3, POU2AF1, CCR7). This study suggests that clinically relevant HARS can be predicted with genes examined in the peripheral blood of baboons within the first 2 days of irradiation.

In addition to the studies discussed above, there are a large number of studies using blood samples from healthy volunteers irradiated ex vivo and samples collected from cancer patients exposed to myeloablative doses of total-body irradiation (150 cGy per fraction from a linear accelerator at a dose rate of 20 cGy/min).[91] In another study, radiation doses of 2 Gy per fraction twice a day for 3 days (a total of 12 Gy) were used.[97] In this study, eight biodosimetry genes (ACTA2, BBC3, CCNG1, CDKN1A, GADD45A, MDK, SERPINE1, Tnfrsf10b) were identified in cancer patients. These studies demonstrate the effects of irradiation on various genes and potential importance of gene profiling for predicting dose of radiation exposure.[98],[99],[100]

Usually, studies of radiation exposure use external total-or partial-body irradiation with X-ray or γ-photon sources. Alternative, but clearly essential models of radiation exposure using different types of radiation sources, for example, α-and β-emitting radionuclides, are also important for studying changes to gene expression profile. Genomic microarray analysis using mice exposed to 137CsCl demonstrated modulation of various genes depending on the time of isotope administration. A comparison of gene expression using a single 2.8 Gy external radiation exposure and accumulated total-body dose of 2.8 Gy emitted internally from 137CsCl administration demonstrated a substantial increase in the gene expression profile with the internal exposure.[101] Since internalized radionuclides are relevant in the management of radiological casualties, such genomic biodosimetry is highly relevant and offers significant potential.

It is also important to study the effects of the dose rate in addition to studying the effects of the level of radiation exposure and types of radiation exposure on gene expression. One study was conducted using the murine animal model and human blood ex vivo model with acute (1.03 Gy/min) or a low-dose-rate (3.1 mGy/min) exposure. These two dose rate exposure types yielded two different gene expression profiles.[102],[103]

mRNAs play a fundamental, all important role in gene expression. Being involved in transmitting genetic information from DNA, mRNAs serve to specify protein expression in the host. It has been demonstrated that a large number of mRNAs are significantly induced in host cells 24-h post-irradiation and such induction lasts through 72-h post-exposure.[104] Results of such study suggest that mRNA expression may provide estimates of radiation exposure and with additional refinements, rough estimates of absorbed radiation doses.[105]

mRNA levels in blood samples from mice exposed to 0.5–10 Gy total-body-radiation collected at 12 h to 7 days post-exposure were reported to be predictive of radiation dose. CDKN1A was found to be the strongest predictor of radiation dose assessment.[106] In another study, using blood samples from pediatric malignancy patients after 2 Gy exposure, several radioresponsive genes were identified and that correlated with findings in the murine model.[97]

Several cytokine mRNAs have been reported for mice exposed to γ-radiation.[35],[107],[108] Further investigation with mRNAs as biomarkers is promising since there are a large number of targets and a wide range of conditions that can make such signaling available.

Micro-RNA

The noncoding RNAs comprise various classes of RNA transcripts that are not transcribed or translated into proteins. They play important roles in regulating the gene transcription, stability of transcripts, and translation into proteins.[109] Micro-RNA (miRNAs) are a conserved class of short, typically 19–22 nucleotides long, noncoding, regulatory RNAs that usually control gene expression by inducing mRNA cleavage or inhibiting translation by base pairing to partially complementary sequences at the posttranscriptional level. During the last three decades, miRNA has been well-established as valuable regulators of gene expression. The evaluation of miRNAs is simple, and miRNA has the potential to be useful diagnostic, prognostic, and predictive biomarkers. Recently, miRNAs have gathered interest as possible biomarkers of radiation dose assessment and extent of injury as a result of radiation exposure.[6],[110] Preclinical investigations in murine and NHP models have demonstrated that serum miRNAs can be used to discriminate pre- and post-irradiation conditions and predict the biological effects of high-dose radiation exposure. Recent studies have suggested that the presence of miRNAs in biofluids (blood or urine) can be valuable for its validation as a biomarker.[111] There are several advantages for using miRNAs as biomarkers for radiation injury: they are evolutionarily conserved across species, relatively stable due to their small size and location in the exosome (those found in the serum are inherently stable), stable in formalin-fixed tissues, and their expression is altered in response to tissue injury and is tissue specific. Further, miRNAs are amenable to and can be scanned using high throughput platforms and levels are reproducible in individuals of the same species. There are several studies during the last 5 years using rodent and NHP models suggesting that miRNAs can predict both the extent of radiation injury and outcome for survival/mortality.[2],[112],[113],[114],[115],[116],[117],[118] It has also been extensively investigated in cancer patients undergoing radiotherapy.[119],[120] Several studies have reported a panel of serum miRNAs that were differentially expressed in response to total-body irradiation in different strains of mice.[5] These studies suggest that the miRNA-based assays are robust and reproducible. In addition, these results demonstrate that the serum miRNAs associated with irradiation quickly return to normal range if a radiation countermeasure is used prior to irradiation,[116],[117] suggesting perhaps, that the serum miRNA signature may not only be helpful in radiation exposure dose assessment but also helpful in the development of radiation countermeasures, specifically in dose conversion from animals to humans under the US FDA Animal Rule. However, it is not always easy to translate the findings achieved in a murine model into clinical practice. Since it is impracticable to test the efficacy of miRNA in humans, specifically against lethal doses of radiation, studies have been conducted in NHPs (rhesus macaque and baboons), an experimental model that closely mimics the human condition.[112],[114],[121] Several miRNAs have been shown to be either up-or-down-regulated in response to irradiation.[112] A few miRNAs have also been linked to mortality of the animals. In a recent study using baboons exposed to 60Co γ-radiation (2.5 or 5 Gy equivalent total-body irradiation, dose rate 8 cGy/min or 32 cGy/min), persistent changes in mRNA and miRNA have been shown.[121] Even after several months after the baboons were irradiated, consistent changes in the levels of 21 miRNAs were observed. Specifically, miR-212 associated with radiosensitivity and immunomodulation was 48–77-fold up-regulated over the entire study period.[121]

In brief, miRNAs are now well-known to be involved in the radiation response, and they may serve as promising predictive and prognostic biomarkers for radiation exposure. It may serve as valuable tools in the future radiation dose assessment for radiation-exposed victims and triage.

Long noncoding RNA

Interest in the noncoding genes has grown significantly over the past few years due to the identification of lncRNA transcription. lncRNAs are >200 nucleotides in length with no protein-coding potential.[122] These are similar to mRNA and are transcribed by RNA polymerase II, polyadenylated and are frequently spliced. Usually, they have fewer exons than coding genes. lncRNAs are intricately regulated and restricted to specific cell types. Now, the lncRNAs contains tens of thousands of genes expressed in differentiated tissues, and the number of lncRNA genes outnumbers protein-coding genes with more than 90% having no peptide products.[123] Based on their proximally adjacent protein-coding genes, lncRNAs are grouped into five classes: Sense, anti-sense, bidirectional, intronic, and intergenic. Several lncRNAs have been identified and proposed as biomarkers for DNA damage, environmental stressors, cancer, and radiation injury. With regard to radiation injury, several lncRNAs respond to irradiation in a time and dose-dependent manner.[124],[125]


  Microbiota Top


Animals and humans are hosts to 10–100 trillion colonizing microbes, a majority of which live in the intestines and with sizable fractions excreted with the feces after irradiation. Several studies using various animal models have demonstrated altered intestinal microbiota in the feces, which suggests that these microbes and their intestinal microenvironment are sensitive to total-body or partial-body irradiation.[126],[127] Such microbes in feces can serve as biomarkers of radiation injury, but these observed changes in intestinal microbes are temporary and short-lived. To be a dependable biomarker for radiation injury, biologically-significant changes needs be sustained and long-lasting. Initial studies relied on the cultivation of fecal bacteria followed by identification and classification of microbiota. Unfortunately, most of the time, bacteria colonizing the gut are not easily cultivatable ex vivo. In recent studies with such microbes, high throughput molecular approach based on species-specific gene sequences has been used. Each bacterial species has a definitive number of genomic copies for the 16S rRNA gene, the numerical abundance of 16S copies in a fecal sample may provide valuable information regarding the relative quantity of that microbe.

The analysis of intestinal microbiota represents a novel approach in attempting to develop radiation exposure-specific biomarkers that can supplement chromosome aberrational analysis or other conventional biomarkers and enhance the range and accuracy of biological dose assessments. In a recent study with Wistar rats, 16S rRNA levels were shown to respond to single and multiple fraction total-body irradiation (10 or 18 Gy, X-ray). Levels of 16S rRNA corresponding to 12 members of Bacteroidales, Lactobacillaceae, and Streptococcaceae increased after radiation exposure; 47 Clostridiaceae members decreased, and 98 Clostridiaceae and Peptostreptococcaceae members remained unchanged.[127] Various altered floras are common in both human and rat feces. Intestinal microbiota-derived metabolomics species have also been reported in blood plasma. The liver and plasma metabolites and lipids profiling of samples extracted 24 h after irradiation with 0, 2, 4, 6, 8, or 10.4 Gy of C57BL/6 mice (n = 12/group) identified 37 compounds whose concentrations correlated well with radiation dose. Levels of pyrimidine positively correlated, and gut tryptophan metabolism associated with gut microbiota negatively correlated with radiation dose. These may turn out to be a potential biomarker for radiation damage.[128]


  Conclusion and Future Direction Top


Several biomarkers have been validated/qualified by the US FDA for indications not related to radiation injury: to date, there are no FDA qualified biomarkers for radiation injury. There are several reasons for such a gap; the major one being the fact that it is unethical to knowingly and intentionally expose humans to IR without a medical purpose. In addition, the process of determining biomarkers for radiation injury through the US FDA Animal Rule is quite slow and technically challenging. In addition, if a radiological/nuclear attack or accident were to occur, medical care providers would concentrate on triaging and treating victims, and they would not have the time or facility for collecting and processing samples from radiation-exposed victims for biomarker identification and validation/qualification. Furthermore, biomarker assessment in partial-body exposure in addition to total-body is needed. Similarly, biomarkers for combined injury also need to be explored as biomarkers for each for these situations (whole-body, partial-body, and combined injury) will be required. The wound or burn effect may complicate and alter biodosimetric assessments. It is not possible to have a universal biomarker for different radiation exposure scenarios that would be useful under different situations. Scientists in the radiation dosimetry field have reached a consensus that there is no perfect biomarker to assess absorbed radiation dose or associated injury under all conditions of exposure: a combination of complementary methods is clearly needed. As stated above, the dicentric assay is considered the gold standard among all cytogenetic techniques. The development of high throughput automatic platforms for supporting cytogenetic assays has been recognized as an urgent need for using cytogenetic methods for large scale screening during any mass casualty scenario. Another effort to make the dicentric assay more useful and technically amenable for a mass casualty scenario is to establish an international network for scoring dicentric images.

Intestinal microbiota analysis by the evaluation of 16S rRNA from feces by high-throughput techniques such as microarray and polymerase chain reaction is a noninvasive technique and provides sustained levels of signal that is increased several folds following exposure to radiation and may serve as novel biomarkers of radiation exposure.

Metabolomics is a highly promising area in the field of radiation biodosimetry that involves noninvasive measurement of radiation-induced changes in the body. It is a powerful technique to study post-irradiation changes. This technology has been used to identify radiation-induced metabolic changes after external exposure as well as after internal radiation exposure with radionuclides. Several promising metabolomics biomarkers have been identified for radiation injury using the different type of samples such as serum, plasma, urine, and tissues; however, identified biomarkers need further validation using different types of radiation, various animal models, and human volunteers.[3]

In addition to the techniques and parameters discussed above, monitoring various clinical signs and symptoms associated with prodromal phase such as nausea, vomiting, diarrhea, and hypertension provide a useful clinical-based approach for determining general levels of radiation exposure. In this regard, the time to emesis after radiation exposure has been demonstrated to correlate with exposure dose.[129]

Acknowledgments

The opinions or assertions contained herein are the private views of the authors and are not necessarily those of the Armed Forces Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, or the Department of Defense. Mention of specific therapeutic agents does not constitute endorsement by the U.S. Department of Defense, and trade names are used only for the purpose of clarification. We apologize to those having contributed substantially to the topics discussed herein that we were unable to cite because of space constraints. We would also like to acknowledge our anonymous reviewers who have helped us to improve the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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