Evan L. Pannkuk

Global Metabolomic Identification of Long-Term Dose-Dependent Urinary Biomarkers in Nonhuman Primates Exposed to Ionizing Radiation

Due to concerns surrounding potential large-scale radiological events, there is a need to determine robust radiation signatures for the rapid identification of exposed individuals, which can then be used to guide the development of compact field deployable instruments to assess individual dose. Metabolomics provides a technology to process easily accessible biofluids and determine rigorous quantitative radiation biomarkers with mass spectrometry (MS) platforms. While multiple studies have utilized murine models to determine radiation biomarkers, limited studies have profiled nonhuman primate (NHP) metabolic radiation signatures. In addition, these studies have concentrated on short-term biomarkers (i.e., <72 h). The current study addresses the need for biomarkers beyond 72 h using a NHP model. Urine samples were collected at 7 days postirradiation (2, 4, 6, 7 and 10 Gy) and processed with ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight (QTOF) MS, acquiring global metabolomic radiation signatures. Multivariate data analysis revealed clear separation between control and irradiated groups. Thirteen biomarkers exhibiting a dose response were validated with tandem MS. There was significantly higher excretion of l-carnitine, l-acetylcarnitine, xanthine and xanthosine in males versus females. Metabolites validated in this study suggest perturbation of several pathways including fatty acid β oxidation, tryptophan metabolism, purine catabolism, taurine metabolism and steroid hormone biosynthesis. In this novel study we detected long-term biomarkers in a NHP model after exposure to radiation and demonstrate differences between sexes using UPLC-QTOF-MS-based metabolomics technology.

Metabolomic applications in radiation biodosimetry: exploring radiation effects through small molecules.

Abstract Purpose: Exposure of the general population to ionizing radiation has increased in the past decades, primarily due to long distance travel and medical procedures. On the other hand, accidental exposures, nuclear accidents, and elevated threats of terrorism with the potential detonation of a radiological dispersal device or improvised nuclear device in a major city, all have led to increased needs for rapid biodosimetry and assessment of exposure to different radiation qualities and scenarios. Metabolomics, the qualitative and quantitative assessment of small molecules in a given biological specimen, has emerged as a promising technology to allow for rapid determination of an individual’s exposure level and metabolic phenotype. Advancements in mass spectrometry techniques have led to untargeted (discovery phase, global assessment) and targeted (quantitative phase) methods not only to identify biomarkers of radiation exposure, but also to assess general perturbations of metabolism with potential long-term consequences, such as cancer, cardiovascular, and pulmonary disease. Conclusions: Metabolomics of radiation exposure has provided a highly informative snapshot of metabolic dysregulation. Biomarkers in easily accessible biofluids and biospecimens (urine, blood, saliva, sebum, fecal material) from mouse, rat, and minipig models, to non-human primates and humans have provided the basis for determination of a radiation signature to assess the need for medical intervention. Here we provide a comprehensive description of the current status of radiation metabolomic studies for the purpose of rapid high-throughput radiation biodosimetry in easily accessible biofluids and discuss future directions of radiation metabolomics research.

Implications of genotypic differences in the generation of a urinary metabolomics radiation signature.

The increased threat of radiological terrorism and accidental nuclear exposures, together with increased usage of radiation-based medical procedures, has made necessary the development of minimally invasive methods for rapid identification of exposed individuals. Genetically predisposed radiosensitive individuals comprise a significant number of the population and require specialized attention and treatments after such events. Metabolomics, the assessment of the collective small molecule content in a given biofluid or tissue, has proven effective in the rapid identification of radiation biomarkers and metabolic perturbations. To investigate how the genotypic background may alter the ionizing radiation (IR) signature, we analyzed urine from Parp1(-/-) mice, as a model radiosensitive genotype, exposed to IR by utilizing the analytical power of liquid chromatography coupled with mass spectrometry (LC-MS), as urine has been thoroughly investigated in wild type (WT) mice in previous studies from our laboratory. Samples were collected at days one and three after irradiation, time points that are important for the early and efficient triage of exposed individuals. Time-dependent perturbations in metabolites were observed in the tricarboxylic acid pathway (TCA). Other differentially excreted metabolites included amino acids and metabolites associated with dysregulation of energy metabolism pathways. Time-dependent apoptotic pathway activation between WT and mutant mice following IR exposure may explain the altered excretion patterns, although the origin of the metabolites remains to be determined. This first metabolomics study in urine from radiation exposed genetic mutant animal models provides evidence that this technology can be used to dissect the effects of genotoxic agents on metabolism by assessing easily accessible biofluids and identify biomarkers of radiation exposure. Applications of metabolomics could be incorporated in the future to further elucidate the effects of IR on the metabolism of Parp1(-/-) genotype by assessing individual tissues.

Targeted metabolomics of nonhuman primate serum after exposure to ionizing radiation: potential tools for high-throughput biodosimetry

There is a need for research to rapidly determine an individuals absorbed dose and its potential health effects after a potential radiological or nuclear event that could expose large portions of a population to ionizing radiation (IR). Studies on biomarker identification after radiation exposure could aid in biodosimetry, identifying individual dose absorbed, as well as biologic response, and administering immediate and proper medical care. Metabolomics on easily accessible biofluids is an emerging field with potential for high-throughput biodosimetry. While tremendous effort has been put into obtaining discovery based global radiation signatures from a number of biofluids and model organisms, quantitative targeted analysis on a subset of known radiation biomarkers is required to develop an optimized panel of biomarkers for future clinical applications. The current study analyzes levels of several known broad chemical groups (acylcarnitines, amino acids, phosphatidylcholines, and biogenic amines) affected by IR in serum from nonhuman primates (NHP) 7 days after exposure through multiple reaction monitoring (MRM) analysis with a triple quadrupole mass spectrometry (MS) platform. We identified several novel metabolites affected by IR exposure through univariate and unsupervised multivariate analyses. Levels of acylcarnitines, amino acids, and phospholipids were perturbed indicating altered protein metabolism, fatty acid β-oxidation, and inflammation. Fold changes in carnitine and short-chain acylcarnitines (acetylcarnitine, propionylcarnitine, butyrylcarnitine, and valerylcarnitine) complement previous global radiation signatures on NHP; notably, the levels of change were lower than previously observed in urine. Decreased levels of glutamate, citrulline, and arginine after IR are biomarkers indicating gastrointestinal syndrome and perturbations to the urea cycle. Sex differences were also assessed and were more prevalent in circulating acylcarnitines and phospholipids after IR exposure. These biomarkers may be combined with previously described compounds from DNA damage to develop a defined metabolomic biodosimetry panel to be analyzed by MS platforms, which are increasingly available in clinical laboratories.

Gas Chromatography/Mass Spectrometry Metabolomics of Urine and Serum from Nonhuman Primates Exposed to Ionizing Radiation: Impacts on the Tricarboxylic Acid Cycle and Protein Metabolism

Ionizing radiation (IR) directly damages cells and tissues or indirectly damages them through reactive free radicals that may lead to longer term adverse sequelae such as cancers, persistent inflammation, or possible death. Potential exposures include nuclear reactor accidents, improper disposal of equipment containing radioactive materials or medical errors, and terrorist attacks. Metabolomics (comprehensive analysis of compounds <1 kDa) by mass spectrometry (MS) has been proposed as a tool for high-throughput biodosimetry and rapid assessment of exposed dose and triage needed. While multiple studies have been dedicated to radiation biomarker discovery, many have utilized liquid chromatography (LC) MS platforms that may not detect particular compounds (e.g., small carboxylic acids or isomers) that complementary analytical tools, such as gas chromatography (GC) time-of-flight (TOF) MS, are ideal for. The current study uses global GC-TOF-MS metabolomics to complement previous LC-MS analyses on nonhuman primate biofluids (urine and serum) 7 days after exposure to 2, 4, 6, 7, and 10 Gy IR. Multivariate data analysis was used to visualize differences between control and IR exposed groups. Univariate analysis was used to determine a combined 26 biomarkers in urine and serum that significantly changed after exposure to IR. We found several metabolites involved in tricarboxylic acid cycle function, amino acid metabolism, and host microbiota that were not previously detected by global and targeted LC-MS studies.

Alterations in Cell Motility, Proliferation, and Metabolism in Novel Models of Acquired Temozolomide Resistant Glioblastoma

Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ in vitro, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the “go” (8MGBA-TMZres) or “grow” (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.

Nonhuman Primates with Acute Radiation Syndrome: Results from a Global Serum Metabolomics Study after 7.2 Gy Total-Body Irradiation

Threats of nuclear terrorism coupled with potential unintentional ionizing radiation exposures have necessitated the need for large-scale response efforts of such events, including high-throughput biodosimetry for medical triage. Global metabolomics utilizing mass spectrometry (MS) platforms has proven an ideal tool for generating large compound databases with relative quantification and structural information in a short amount of time. Determining metabolite panels for biodosimetry requires experimentation to evaluate the many factors associated with compound concentrations in biofluids after radiation exposures, including temporal changes, pre-existing conditions, dietary intake, partial- vs. total-body irradiation (TBI), among others. Here, we utilize a nonhuman primate (NHP) model and identify metabolites perturbed in serum after 7.2 Gy TBI without supportive care [LD70/60, hematologic (hematopoietic) acute radiation syndrome (HARS) level H3] at 24, 36, 48 and 96 h compared to preirradiation samples with an ultra-performance liquid chromatography quadrupole time-of-flight (UPLC-QTOF) MS platform. Additionally, we document changes in cytokine levels. Temporal changes observed in serum carnitine, acylcarnitines, amino acids, lipids, deaminated purines and increases in pro-inflammatory cytokines indicate clear metabolic dysfunction after radiation exposure. Multivariate data analysis shows distinct separation from preirradiation groups and receiver operator characteristic curve analysis indicates high specificity and sensitivity based on area under the curve at all time points after 7.2 Gy irradiation. Finally, a comparison to a 6.5 Gy (LD50/60, HARS level H2) cohort after 24 h postirradiation revealed distinctly increased separations from the 7.2 Gy cohort based on multivariate data models and higher compound fold changes. These results highlight the utility of MS platforms to differentiate time and absorbed dose after a potential radiation exposure that may aid in assigning specific medical interventions and contribute as additional biodosimetry tools.

Lipidomic Signatures of Nonhuman Primates with Radiation-Induced Hematopoietic Syndrome

Concern over potential exposures of ionizing radiation (IR) to large populations has emphasized the need for rapid and reliable methods of biodosimetry to determine absorbed dose and required triage. Lipidomics has emerged as a powerful technique for large-scale lipid identification and quantification. Indirect effects from IR exposure generate reactive oxygen species (ROS) through water hydrolysis and may subsequently damage cellular lipids. Thus, rapid identification of specific affected lipid molecules represents possible targets for biodosimetry. The current study addresses temporal changes in the serum lipidome from 4 h to 28 d in nonhuman primates (NHPs) with radiation-induced hematopoietic syndrome (6.5 Gy exposure, LD50/60). Statistical analyses revealed a highly dynamic temporal response in the serum lipidome after IR exposure. Marked lipidomic perturbations occurred within 24 h post-irradiation along with increases in cytokine levels and C-reactive protein. Decreases were observed in di- and triacylglycerides, sphingomyelins (SMs), lysophosphatidylcholines (LysoPCs), and esterified sterols. Conversely, free fatty acids and monoacylglycerides significantly increased. Decreased levels of SMs and increased levels of LysoPCs may be important markers for biodosimetry ~2 d–3 d post-irradiation. The biphasic and dynamic response to the serum lipidome post-irradiation emphasize the importance of determining the temporal long-term response of possible radiation markers.

A Serum Small Molecule Biosignature of Radiation Exposure from Total Body Irradiated Patients

The potential for radiological accidents and nuclear terrorism has increased the need for the development of new rapid biodosimetry methods. In addition, in a clinical setting the issue of an individuals radiosensitivity should be taken into consideration during radiotherapy. We utilized metabolomics and lipidomics to investigate changes of metabolites in serum samples following exposure to total body ionizing radiation in humans. Serum was collected prior to irradiation, at 3-8 h after a single dose of 1.25-2 Gy, and at 24 h with a total delivered dose of 2-3.75 Gy. Metabolomics revealed perturbations in glycerophosphocholine, phenylalanine, ubiquinone Q2, and oxalic acid. Alterations were observed in circulating levels of lipids from monoacylglycerol, triacylglycerol, phosphatidylcholine, and phosphatidylglycerol lipid classes. Polyunsaturated fatty acids were some of the most dysregulated lipids, with increased levels linked to proinflammatory processes. A targeted metabolomics approach for eicosanoids was also employed. The results showed a rapid response for proinflammatory eicosanoids, with a dampening of the signal at the later time point. Sex differences were observed in the markers from the untargeted approach but not the targeted method. The ability to identify and quantify small molecules in blood can therefore be utilized to monitor radiation exposure in human populations.