Impact of Partial Body Shielding from Very High Dose Rates on Untargeted Metabolomics in Biodosimetry.

A realistic exposure to ionizing radiation (IR) from an improvised nuclear device will likely include individuals who are partially shielded from the initial blast delivered at a very high dose rate (VHDR). As different tissues have varying levels of radiosensitivity, e.g., hematopoietic vs gastrointestinal tissues, the effects of shielding on radiation biomarkers need to be addressed. Here, we explore how biofluid (urine and serum) metabolite signatures from male and female C57BL/6 mice exposed to VHDR (5-10 Gy/s) total body irradiation (TBI, 0, 4, and 8 Gy) compare to individuals exposed to partial body irradiation (PBI) (lower body irradiated [LBI] or upper body irradiated [UBI] at an 8 Gy dose) using a data-independent acquisition untargeted metabolomics approach. Although sex differences were observed in the spatial groupings of urine signatures from TBI and PBI mice, a metabolite signature (N6,N6,N6-trimethyllysine, carnitine, propionylcarnitine, hexosamine-valine-isoleucine, taurine, and creatine) previously developed from variable dose rate experiments was able to identify individuals with high sensitivity and specificity, irrespective of radiation shielding. A panel of serum metabolites composed from previous untargeted studies on nonhuman primates had excellent performance for separating irradiated cohorts; however, a multiomic approach to complement the metabolome could increase dose estimation confidence intervals. Overall, these results support the inclusion of small-molecule markers in biodosimetry assays without substantial interference from the upper or lower body shielding.

Radiation therapy promotes unsaturated fatty acids to maintain survival of glioblastoma.

Radiation therapy (RT) is essential for the management of glioblastoma (GBM). However, GBM frequently relapses within the irradiated margins, thus suggesting that RT might stimulate mechanisms of resistance that limits its efficacy. GBM is recognized for its metabolic plasticity, but whether RT-induced resistance relies on metabolic adaptation remains unclear. Here, we show in vitro and in vivo that irradiated GBM tumors switch their metabolic program to accumulate lipids, especially unsaturated fatty acids. This resulted in an increased formation of lipid droplets to prevent endoplasmic reticulum (ER) stress. The reduction of lipid accumulation with genetic suppression and pharmacological inhibition of the fatty acid synthase (FASN), one of the main lipogenic enzymes, leads to mitochondrial dysfunction and increased apoptosis of irradiated GBM cells. Combination of FASN inhibition with focal RT improved the median survival of GBM-bearing mice. Supporting the translational value of these findings, retrospective analysis of the GLASS consortium dataset of matched GBM patients revealed an enrichment in lipid metabolism signature in recurrent GBM compared to primary. Overall, these results demonstrate that RT drives GBM resistance by generating a lipogenic environment permissive to GBM survival. Targeting lipid metabolism might be required to develop more effective anti-GBM strategies.

Variable Dose Rates in Realistic Radiation Exposures: Effects on Small Molecule Markers of Ionizing Radiation in the Murine Model.

Novel biodosimetry assays for use in preparedness and response to potential malicious attacks or nuclear accidents would ideally provide accurate dose reconstruction independent of the idiosyncrasies of a complex exposure to ionizing radiation. Complex exposures will consist of dose rates spanning the low dose rates (LDR) to very high-dose rates (VHDR) that need to be tested for assay validation. Here, we investigate how a range of relevant dose rates affect metabolomic dose reconstruction at potentially lethal radiation exposures (8 Gy in mice) from an initial blast or subsequent fallout exposures compared to zero or sublethal exposures (0 or 3 Gy in mice) in the first 2 days, which corresponds to an integral time individuals will reach medical facilities after a radiological emergency. Biofluids (urine and serum) were collected from both male and female 9-10-week-old C57BL/6 mice at 1 and 2 days postirradiation (total doses of 0, 3 or 8 Gy) after a VHDR of 7 Gy/s. Additionally, samples were collected after a 2-day exposure consisting of a declining dose rate (1 to 0.004 Gy/min) recapitulating the 7:10 rule-of-thumb time dependency of nuclear fallout. Overall similar perturbations were observed in both urine and serum metabolite concentrations irrespective of sex or dose rate, with the exception of xanthurenic acid in urine (female specific) and taurine in serum (VHDR specific). In urine, we developed identical multiplex metabolite panels (N6, N6,N6-trimethyllysine, carnitine, propionylcarnitine, hexosamine-valine-isoleucine, and taurine) that could identify individuals receiving potentially lethal levels of radiation from the zero or sublethal cohorts with excellent sensitivity and specificity, with creatine increasing model performance at day 1. In serum, individuals receiving a 3 or 8 Gy exposure could be identified from their pre-irradiation samples with excellent sensitivity and specificity, however, due to a lower dose response the 3 vs. 8 Gy groups could not be distinguished from each other. Together with previous results, these data indicate that dose-rate-independent small molecule fingerprints have potential in novel biodosimetry assays.

Quantitative proteomic analytic approaches to identify metabolic changes in the medial prefrontal cortex of rats exposed to space radiation.

NASA's planned mission to Mars will result in astronauts being exposed to ∼350 mSv/yr of Galactic Cosmic Radiation (GCR). A growing body of data from ground-based experiments indicates that exposure to space radiation doses (approximating those that astronauts will be exposed to on a mission to Mars) impairs a variety of cognitive processes, including cognitive flexibility tasks. Some studies report that 33% of individuals may experience severe cognitive impairment. Translating the results from ground-based rodent studies into tangible risk estimates for astronauts is an enormous challenge, but it would be germane for NASA to use the vast body of data from the rodent studies to start developing appropriate countermeasures, in the expectation that some level of space radiation (SR) -induced cognitive impairment could occur in astronauts. While some targeted studies have reported radiation-induced changes in the neurotransmission properties and/or increased neuroinflammation within space radiation exposed brains, there remains little information that can be used to start the development of a mechanism-based countermeasure strategy. In this study, we have employed a robust label-free mass spectrometry (MS) -based untargeted quantitative proteomic profiling approach to characterize the composition of the medial prefrontal cortex (mPFC) proteome in rats that have been exposed to 15 cGy of 600 MeV/n28Si ions. A variety of analytical techniques were used to mine the generated expression data, which in such studies is typically hampered by low and variable sample size. We have identified several pathways and proteins whose expression alters as a result of space radiation exposure, including decreased mitochondrial function, and a further subset of proteins differs in rats that have a high level of cognitive performance after SR exposure in comparison with those that have low performance levels. While this study has provided further insight into how SR impacts upon neurophysiology, and what adaptive responses can be invoked to prevent the emergence of SR-induced cognitive impairment, the main objective of this paper is to outline strategies that can be used by others to analyze sub-optimal data sets and to identify new information.

Application of radiation omics in the development of adverse outcome pathway networks: an example of radiation-induced cardiovascular disease.

BACKGROUND: Epidemiological studies have indicated that exposure of the heart to doses of ionizing radiation as low as 0.5 Gy increases the risk of cardiac morbidity and mortality with a latency period of decades. The damaging effects of radiation to myocardial and endothelial structures and functions have been confirmed radiobiologically at high dose, but much less are known at low dose. Integration of radiation biology and epidemiology data is a recommended approach to improve the radiation risk assessment process. The adverse outcome pathway (AOP) framework offers a comprehensive tool to compile and translate mechanistic information into pathological endpoints which may be relevant for risk assessment at the different levels of a biological system. Omics technologies enable the generation of large volumes of biological data at various levels of complexity, from molecular pathways to functional organisms. Given the quality and quantity of available data across levels of biology, omics data can be attractive sources of information for use within the AOP framework. It is anticipated that radiation omics studies could improve our understanding of the molecular mechanisms behind the adverse effects of radiation on the cardiovascular system. In this review, we explored the available omics studies on radiation-induced cardiovascular disease (CVD) and their applicability to the proposed AOP for CVD. RESULTS: The results of 80 omics studies published on radiation-induced CVD over the past 20 years have been discussed in the context of the AOP of CVD proposed by Chauhan et al. Most of the available omics data on radiation-induced CVD are from proteomics, transcriptomics, and metabolomics, whereas few datasets were available from epigenomics and multi-omics. The omics data presented here show great promise in providing information for several key events (KEs) of the proposed AOP of CVD, particularly oxidative stress, alterations of energy metabolism, extracellular matrix (ECM), and vascular remodeling. CONCLUSIONS: The omics data presented here shows promise to inform the various levels of the proposed AOP of CVD. However, the data highlight the urgent need of designing omics studies to address the knowledge gap concerning different radiation scenarios, time after exposure, and experimental models. This review presents the evidence to build a qualitative omics-informed AOP and provides views on the potential benefits and challenges in using omics data to assess risk-related outcomes.

Biofluid Metabolomics and Lipidomics of Mice Exposed to External Very High-Dose Rate Radiation.

High-throughput biodosimetry methods to determine exposure to ionizing radiation (IR) that can also be easily scaled to multiple testing sites in emergency situations are needed in the event of malicious attacks or nuclear accidents that may involve a substantial number of civilians. In the event of an improvised nuclear device (IND), a complex IR exposure will have a very high-dose rate (VHDR) component from an initial blast. We have previously addressed low-dose rate (LDR, ≤1 Gy/day) exposures from internal emitters on biofluid small molecule signatures, but further research on the VHDR component of the initial blast is required. Here, we exposed 8- to 10-week-old male C57BL/6 mice to an acute dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a VHDR of 7 Gy/s, collected urine and serum at 1 and 7 d, then compared the metabolite signatures using either untargeted (urine) or targeted (serum) approaches with liquid chromatography mass spectrometry platforms. A Random Forest classification approach showed strikingly similar changes in urinary signatures at 1 d post-irradiation with VHDR samples grouping closer to control samples at 7 d. Identical metabolite panels (carnitine, trigonelline, xanthurenic acid, N6,N6,N6-trimethyllysine, spermine, and hexosamine-valine-isoleucine-OH) could differentiate IR exposed individuals with high sensitivity and specificity (area under the receiver operating characteristic (AUROC) curves 0.89-1.00) irrespective of dose rate at both days. For serum, the top 25 significant lipids affected by IR exposure showed slightly higher perturbations at 0.7 Gy/min vs. 7 Gy/s; however, identical panels showed excellent sensitivity and specificity at 1 d (three hexosylceramides (16:0), (18:0), (24:0), sphingomyelin [26:1], lysophosphatidylethanolamine [22:1]). Mice could not be differentiated from control samples at 7 d for a 3 Gy exposure based on serum lipid signatures. As with LDR exposures, we found that identical biofluid small molecule signatures can identify IR exposed individuals irrespective of dose rate, which shows promise for more universal applications of metabolomics for biodosimetry.

Small Molecule Signatures of Mice Lacking T-cell p38 Alternate Activation, a Model for Immunosuppression Conditions, after Total-Body Irradiation.

Several diagnostic biodosimetry tools have been in development that may aid in radiological/nuclear emergency responses. Of these, correlating changes in non-invasive biofluid small-molecule signatures to tissue damage from ionizing radiation exposure show promise for inclusion in predictive biodosimetry models. Integral to dose reconstruction has been determining how genotypic variation in the general population will affect model performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway [p38αßY323F, double knock-in (DKI) mice] to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. We exposed adult male DKI mice (8-10 weeks old) to 2 and 7 Gy in parallel with wild-type mice and assessed perturbations in urine (days 1, 3, 7) and serum (day 1) using a global metabolomics approach. A multidimensional scaling plot showed excellent separation of radiation-exposed groups in wild-type mice with slightly dampened responses in DKI mice. Validated metabolite panels were developed for urine [N6,N6,N6-trimethyllysine (TML), N1-acetylspermidine, spermidine, carnitine, acylcarnitine C21H35NO5, aminohippuric acid] and serum [phenylalanine, glutamine, propionylcarnitine, lysophosphatidylcholine (LysoPC 14:0), LysoPC (22:5)] to determine the area under the receiver operating characteristic curve (AUROC). For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤2 Gy vs. 7 Gy mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes after irradiation. Overall, these results suggest immunosuppression should not compromise small molecule multiplex panels used in dose reconstruction for biodosimetry.

Effect of the p38 Mitogen-Activated Protein Kinase Signaling Cascade on Radiation Biodosimetry.

Radiation biodosimetry based on transcriptomic analysis of peripheral blood is a valuable tool to detect radiation exposure after a radiological/nuclear event and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation or immune suppression, can potentially obscure the predictive power of the method. Members of the p38 mitogen-activated protein kinase (MAPK) family respond to pro-inflammatory signals and environmental stresses, whereas genetic ablation of the p38 signaling pathway in mice leads to reduced susceptibility to collagen-induced arthritis and experimental autoimmune encephalomyelitis that model human rheumatoid arthritis and multiple sclerosis, respectively. p38 is normally regulated by the MAP3K-MAP2K pathway in mammalian cells. However, in T cells there is an alternative pathway for p38 activation that plays an important role in antigen-receptor-activated T cells and participates in immune and inflammatory responses. To examine the role of p38 in response to radiation, we used two mouse models expressing either a p38α dominant negative (DN) mutation that globally suppresses p38 signaling or a p38αß double-knock-in (DKI) mutant, which inhibits specifically T-cell receptor activation. We exposed p38 wild-type (p38WT) and mutant male mice to 7 Gy X rays and 24 h later whole blood was isolated subjected to whole-genome microarray and gene ontology analysis. Irradiation of p38WT mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. In contrast, these pathways were significantly underrepresented in p38DN and p38DKI mutant mice, suggesting that p38 attenuation may protect blood cells from the deleterious effects of radiation. Furthermore, radiation exposure in p38 mutant mice resulted in an enrichment of phagocytosis-related pathways, suggesting a role for p38 signaling in restricting phagocytosis of apoptotic cells after irradiation. Finally, despite the significant changes in gene expression, it was still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of p38 status.

Biofluid Metabolomics of Mice Exposed to External Low-Dose Rate Radiation in a Novel Irradiation System, the Variable Dose-Rate External 137Cs Irradiator.

An important component of ionizing radiation (IR) exposure after a radiological incident may include low-dose rate (LDR) exposures either externally or internally, such as from 137Cs deposition. In this study, a novel irradiation system, VAriable Dose-rate External 137Cs irradiatoR (VADER), was used to expose male and female mice to a variable LDR irradiation over a 30 d time span to simulate fall-out-type exposures in addition to biofluid collection from a reference dose rate (0.8 Gy/min). Radiation markers were identified by untargeted metabolomics and random forests. Mice exposed to LDR exposures were successfully identified from control groups based on their urine and serum metabolite profiles. In addition to metabolites commonly perturbed after IR exposure, we identified and validated a novel metabolite (hexosamine-valine-isoleucine-OH) that increased up to 150-fold after LDR and 80-fold after conventional exposures in urine. A multiplex panel consisting of hexosamine-valine-isoleucine-OH with other urinary metabolites (N6,N6,N6-trimethyllysine, carnitine, 1-methylnicotinamide, and α-ketoglutaric acid) achieved robust classification performance using receiver operating characteristic curve analysis, irrespective of the dose rate or sex. These results show that in terms of biodosimetry, dysregulated energy metabolism is associated with IR exposure for both LDR and conventional IR exposures. These mass spectrometry data have been deposited to the NIH data repository via Metabolomics Workbench with study IDs ST001790, ST001791, ST001792, ST001793, and ST001806.

Effects of Low Dose Space Radiation Exposures on the Splenic Metabolome.

Future space missions will include a return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g., microgravity. Immune regulation is known to be impacted by both radiation and spaceflight and it remains to be seen whether prolonged effects that will be encountered in deep space can have an adverse impact on health. In this study, we investigated the effects in the overall metabolism of three different low dose radiation exposures (γ-rays, 16O, and 56Fe) in spleens from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while later responses shifted towards more prominent responses in groups with high energy particle irradiations. Regression analysis showed a positive correlation of the abundance of identified fatty acids with time and a negative association with γ-rays, while the degradation pathway of purines was positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long-term effects on DNA repair and nucleotide pools following radiation exposure.

Metabolomic Profiling for Diagnosis and Prognostication in Surgery: A Scoping Review.

OBJECTIVE: This review assimilates and critically evaluates available literature regarding the use of metabolomic profiling in surgical decision-making. BACKGROUND: Metabolomic profiling is performed by nuclear magnetic resonance spectroscopy or mass spectrometry of biofluids and tissues to quantify biomarkers (ie, sugars, amino acids, and lipids), producing diagnostic and prognostic information that has been applied among patients with cardiovascular disease, inflammatory bowel disease, cancer, and solid organ transplants. METHODS: PubMed was searched from 1995 to 2019 to identify studies investigating metabolomic profiling of surgical patients. Articles were included and assimilated into relevant categories per PRISMA-ScR guidelines. Results were summarized with descriptive analytical methods. RESULTS: Forty-seven studies were included, most of which were retrospective studies with small sample sizes using various combinations of analytic techniques and types of biofluids and tissues. Results suggest that metabolomic profiling has the potential to effectively screen for surgical diseases, suggest diagnoses, and predict outcomes such as postoperative complications and disease recurrence. Major barriers to clinical adoption include a lack of high-level evidence from prospective studies, heterogeneity in study design regarding tissue and biofluid procurement and analytical methods, and the absence of large, multicenter metabolome databases to facilitate systematic investigation of the efficacy, reproducibility, and generalizability of metabolomic profiling diagnoses and prognoses. CONCLUSIONS: Metabolomic profiling research would benefit from standardization of study design and analytic approaches. As technologies improve and knowledge garnered from research accumulates, metabolomic profiling has the potential to provide personalized diagnostic and prognostic information to support surgical decision-making from preoperative to postdischarge phases of care.

Irradiation of the kidneys causes pathologic remodeling in the nontargeted heart: A role for the immune system.

Cardiac disease is a frequent and significant adverse event associated with radiotherapy for cancer. Identifying the underlying mechanism responsible for radiation injury to the heart will allow interventions to be developed. In the present study, we tested if local kidney irradiation results in remodeling of the shielded, nontargeted heart. One kidney, two kidneys, or the total body of male WAG and Dahl SS rats were irradiated with 10 Gy of X-rays. Local kidney irradiation resulted in systemic hypertension, increased BUN, infiltration of T lymphocytes, natural killer cells, and macrophages into the renal cortex and medulla, and renal fibrosis. Local irradiation of kidneys in WAG rats resulted in remodeling in the nontargeted heart after 120 days, manifested by perivascular fibrosis and increased interventricular septal thickness, but was not seen in Dahl SS rats due to a high baseline level of fibrosis in the sham-irradiated animals. Genetic depletion of T cells mitigated the nephropathy after local kidney irradiation, indicating a role for the immune system in mediating this outcome. Local kidney irradiation resulted in a cascade of pro-inflammatory cytokines and low-molecular weight metabolites into the circulation associated with transmission of signals resulting in pathologic remodeling in the nontargeted heart. A new model is proposed whereby radiation-induced cardiac remodeling in susceptible animals is indirect, with lower hemi body organs such as the kidney exporting factors into the circulation that cause remodeling outside of the irradiated field in the shielded, nontargeted heart. This nontargeted effect appears to be mediated, in part, by the immune system.

VADER: a variable dose-rate external 137Cs irradiator for internal emitter and low dose rate studies.

In the long term, 137Cs is probably the most biologically important agent released in many accidental (or malicious) radiation disasters. It can enter the food chain, and be consumed, or, if present in the environment (e.g. from fallout), can provide external irradiation over prolonged times. In either case, due to the high penetration of the energetic γ rays emitted by 137Cs, the individual will be exposed to a low dose rate, uniform, whole body, irradiation. The VADER (VAriable Dose-rate External 137Cs irradiatoR) allows modeling these exposures, bypassing many of the problems inherent in internal emitter studies. Making use of discarded 137Cs brachytherapy seeds, the VADER can provide varying low dose rate irradiations at dose rates of 0.1 to 1.2 Gy/day. The VADER includes a mouse "hotel", designed to allow long term simultaneous residency of up to 15 mice. Two source platters containing ~ 250 mCi each of 137Cs brachytherapy seeds are mounted above and below the "hotel" and can be moved under computer control to provide constant low dose rate or a varying dose rate mimicking 137Cs biokinetics in mouse or man. We present the VADER design and characterization of its performance over 18 months of use.

Effects of Genetic Variation on Urinary Small Molecule Signatures of Mice after Exposure to Ionizing Radiation: A Study of p53 Deficiency.

Due to risks from potential exposures to ionizing radiation (IR), improved radiological countermeasures are required, as well as rapid high-throughput biodosimetry. Genotypic variation in the general population contributes to differences in radiosensitivity that may affect biodosimetry accuracy. Previous studies utilized radiosensitive mutant mouse models (Parp1-/- and Atm-/-) to determine the effects of genotypic deficiency on radiation signatures. Here, we extend this approach by examining changes in the urinary metabolome in a hematopoietic (HP) resistant mouse model (p53-/-) after IR exposure. As p53 is a primary regulator in radiation response and apoptosis, limited hematopoietic stem cell apoptosis leads to reduced mortality at doses of ~8-10 Gy but increased mortality at higher doses (> 15 Gy) due to mitotic catastrophe in gastrointestinal (GI) crypt cells. Urine was collected from mice (wild-type (WT), p53+/-, and p53-/-) pre-irradiation and at 4 and 24 h after total body irradiation (TBI) (WT: 8 and 10 Gy; p53-/-: 10 Gy) for metabolic phenotyping using an ultra-performance liquid chromatography mass spectrometry (UPLC-MS) platform. Minimal differences were detected between unirradiated WT, p53+/-, and p53-/- mice. While similar perturbations were observed for metabolites involved in tryptophan, vitamin B6, and histamine pathways, glycine conjugation, and redox metabolism for WT and p53-/- mice after TBI, an overall dampened response was observed in p53-deficient mice. Despite comparable metabolite patterns between genotypes, differentiation was achieved through receiver operating characteristic curve analysis with high specificity and sensitivity for carnitine, N1-acetylspermidine, and creatine. These studies highlight that both attenuated and dampened metabolic responses due to genetic variability in the general population need to be addressed in biodosimetry frameworks.

Effect of 3,3'-Diindolylmethane on Pulmonary Injury Following Thoracic Irradiation in CBA Mice.

The molecule 3,3'-diindolylmethane (DIM) is small, a major bioactive metabolite of indole-3 carbinol (13C), and a phytochemical compound from cruciferous vegetables released upon exposure to the gut acid environment. DIM is a proposed anti-cancer agent and was previously demonstrated to prevent radiation damage in the bone marrow and the gastrointestinal tract. Here we investigated the effect of DIM on radiation-induced injury to the lung in a murine model through untargeted metabolomics and gene expression studies of select genes. CBA mice were exposed to thoracic irradiation (17.5 Gy). Mice were treated with vehicle or DIM (250 mg kg, subcutaneous injection) on days -1 pre-irradiation through +14 post-irradiation. DIM induced a significant improvement in survival by day 150 post-irradiation. Fibrosis-related gene expression and metabolomics were examined using lung tissue from days 15, 45, 60, 90, and 120 post-irradiation. Our qRT-PCR experiments showed that DIM treatment reduced radiation-induced late expression of collagen Iα and the cell cycle checkpoint proteins p21/waf1 (CDKN1A) and p16ink (CDKN2A). Metabolomic studies of lung tissue demonstrated a significant dampening of radiation-induced changes following DIM treatment. Metabolites associated with pro-inflammatory responses and increased oxidative stress, such as fatty acids, were suppressed by DIM treatment compared to irradiated samples. Together these data suggest that DIM reduces radiation-induced sequelae in the lung.

Metabolomic approaches to study the tumor microenvironment.

Tumors are characterized by metabolic dysregulation, reprogramming, and the presence of metabolites, which can act both as energy mediators and signaling messengers. Measuring the concentration and composition of metabolites in the tumor microenvironment can help to better understand the tumor pathology and might improve therapeutic treatments. Metabolomics can provide a description of the physiological and pathological status, as well as help to identify biomarkers of the disease. Additionally, mass spectrometry-based tissue imaging techniques can show the spatial distribution of metabolites. In this chapter we present protocols for the extraction and analysis of metabolites and lipids, with emphasis on liquid chromatography-mass spectrometry and mass spectrometry imaging.

Quantitation of Urinary Acylcarnitines by DMS-MS/MS Uncovers the Effects of Total Body Irradiation in Cancer Patients.

Acylcarnitines have been identified in human and animal metabolomic-profiling studies as urinary markers of radiation exposure, a result which is consistent with their cytoprotective effects and roles in energy metabolism. In the present work, a rapid method for quantitation of the more abundant acylcarnitines in human urine is developed using a valuable set of samples from cancer patients who received total body irradiation (TBI) at Memorial Sloan Kettering Cancer Center. The method uses solid-phase extraction (SPE) processing followed by differential mobility spectrometry (DMS with ethyl acetate modifier) tandem mass spectrometry (ESI-DMS-MS/MS) with deuterated internal standards. The analyzed human urine samples were collected from 38 individual patients at three time points over 24 h during and after the course of radiation treatment, a design allowing each patient to act as their own control and creatinine normalization. Creatinine-normalized concentrations for nine urinary acylcarnitine (acyl-CN) species are reported. Six acyl-CN species were reduced at the 6 h point. Acetylcarnitine (C2:0-CN) and valerylcarnitine (C5:0-CN) showed recovery at 24 h, but none of the other acyl-CN species showed recovery at that point. Levels of three acyl-CN species were not significantly altered by radiation. This rapid quantitative method for clinical samples covers the short- and medium-chain acylcarnitines and has the flexibility to be expanded to cover additional radiation-linked metabolites. The human data presented here indicates the utility of the current approach as a rapid, quantitative technique with potential applications by the medical community, by space research laboratories concerned with radiation exposure, and by disaster response groups.

Salivary Metabolomics of Total Body Irradiated Nonhuman Primates Reveals Long-Term Normal Tissue Responses to Radiation.

PURPOSE: To identify metabolomic biomarkers of acute radiation exposure in saliva that show time-dependent changes. METHODS AND MATERIALS: Nonhuman primates were exposed to 4 Gy of total body irradiation with γ-rays. Saliva was collected from 7 animals twice before and at days 1, 3, 5, 7, 15, 21, 28, and 60 after irradiation. Profiling was conducted with liquid chromatography time-of-flight mass spectrometry. Multivariate data analysis and potential biomarker identification was conducted through random Forests and the software MetaboAnalyst. Candidate biomarkers were validated through tandem mass spectrometry, and receiver operating characteristic curves were constructed to show the diagnostic ability of the signature over time. RESULTS: Untargeted metabolomic analysis revealed significant and persistent effects up to the 60 days evaluated in this study. Biomarkers spanning primarily amino acids and nucleotides were identified, with a significant number showing long-term responses. Fifteen biomarkers showed high statistical significance in the first week after irradiation and 16 at >7 days after irradiation (false discovery rate-adjusted P < .05). The combination of the biomarkers in a single biosignature was able to accurately show the diagnostic ability of the signature in a binary classifier system with receiver operating characteristic curves. CONCLUSIONS: Radiation can alter the metabolome in saliva, and metabolomics could effectively be used to monitor radiation responses, as a biodosimetry method, in the event of a radiological incident. Saliva metabolomics also has potential relevance in a clinical setting.