Publications by authors named "Albert J Fornace"

204 Publications

GADD45 in Stress Signaling, Cell Cycle Control, and Apoptosis.

Adv Exp Med Biol 2022 ;1360:1-22

Lombardi Comprehensive Cancer Center, Washington, DC, USA.

GADD45 is a gene family consisting of GADD45A, GADD45B, and GADD45G that is often induced by DNA damage and other stress signals associated with growth arrest and apoptosis. Many of these roles are carried out via signaling mediated by p38 mitogen-activated protein kinases (MAPKs). The GADD45 proteins can contribute to p38 activation either by activation of upstream kinase(s) or by direct interaction, as well as suppression of p38 activity in certain cases. In vivo, there are important tissue and cell type specific differences in the roles for GADD45 in MAPK signaling. In addition to being p53-regulated, GADD45A has also been found to contribute to p53 activation via p38. Like other stress and signaling proteins, GADD45 proteins show complex regulation and numerous effectors. More recently, aberrant GADD45 expression has been found in several human cancers, but the mechanisms behind these findings largely remain to be understood.
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http://dx.doi.org/10.1007/978-3-030-94804-7_1DOI Listing
May 2022

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

Radiat Res 2022 Mar 30. Epub 2022 Mar 30.

Center for Radiological Research, Columbia University Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York.

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 p38αβ 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.
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http://dx.doi.org/10.1667/RADE-21-00240.1DOI Listing
March 2022

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

Radiat Res 2022 Mar 4. Epub 2022 Mar 4.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC.

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 [p38αβY323F, 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, 4-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.
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http://dx.doi.org/10.1667/RADE-21-00199.1DOI Listing
March 2022

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow.

Heliyon 2022 Jan 29;8(1):e08691. Epub 2021 Dec 29.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/μm), or high-LET Fe-ions (148 keV/μm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.
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http://dx.doi.org/10.1016/j.heliyon.2021.e08691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8741516PMC
January 2022

Heavy ion space radiation triggers ongoing DNA base damage by downregulating DNA repair pathways.

Life Sci Space Res (Amst) 2020 Nov 7;27:27-32. Epub 2020 Jul 7.

Department of Biochemistry and Molecular & Cellular Biology, Lombardi Comprehensive Cancer Center, Georgetown University, Research Building, Room E518, 3970 Reservoir Rd., NW, Washington, DC 20057, USA. Electronic address:

Long-duration space missions outside low earth orbit will expose astronauts to a cumulative dose of high-energy particle radiation especially to highly damaging heavy ion radiation, which poses considerable risk to astronauts' health. The purpose of the current study was to quantitatively identify oxidatively induced DNA base modifications and assess status of the repair pathways involved in removing the modified bases in mouse intestinal cells after exposure to γ-rays and iron radiation. Mice (C57BL/6J; 6 to 8 weeks; female) were exposed to 0.5 Gy of either γ-rays or iron radiation and control mice were sham-irradiated. Intestinal tissues were collected 2 months after radiation. DNA base lesions were measured using gas chromatography-tandem mass spectrometry with isotope‑dilution. Base excision repair (BER) and nucleotide excision repair (NER) pathways were assessed using PCR and immunoblotting. Effects of iron radiation were compared to γ-rays and sham-irradiated controls. Exposure to iron radiation resulted in significantly higher levels of several DNA base lesions relative to control animals and those exposed to γ radiation. Assessment of BER and NER showed downregulation of pathway factors both at the RNA as well as at the protein levels. Our results not only provide important insight into DNA damage pattern in intestinal cells in response to iron radiation, but they also confirm our previous immunohistochemistry data on oxidatively induced DNA damage. We suggest that downregulation of the BER and NER pathways is contributing to ongoing DNA base damages long time after radiation exposure and has implications for chronic diseases including gastrointestinal diseases after heavy ion radiation exposure during space travel.
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http://dx.doi.org/10.1016/j.lssr.2020.07.001DOI Listing
November 2020

Effects of dietary aspirin on high-LET radiation-induced prostaglandin E2 levels and gastrointestinal tumorigenesis in Apc mice.

Life Sci Space Res (Amst) 2021 Nov 10;31:85-91. Epub 2021 Sep 10.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA; Department of Biochemistry and Molecular & Cellular Biology and Department of Oncology, Georgetown University Medical Center, Washington, DC 20057, USA.

Inevitable exposure to high-LET ionizing radiation (IR) present in galactic cosmic radiation (GCR) could enhance gastrointestinal (GI) cancer incidence among astronauts undertaking deep space exploration and GI-cancer mortality has been predicted to far exceed NASA's limit of < 3% REID (Radiation exposure-induced death) from cancer. Therefore, the development of countermeasure agents against high-LET radiation-induced GI cancer is needed to safeguard astronauts during and after an outer space mission. The cyclooxygenase-2/prostaglandin E2 (COX2/PGE2) mediated activation of pro-inflammatory and oncogenic signaling has been reported to play an important role in persistent inflammation and GI-tumorigenesis after high-LET radiation exposure. Therefore, aspirin, a well-known inhibitor of the COX/PGE2 pathway, was evaluated as a potential countermeasure against Si-induced PGE2 and tumorigenesis in Apc, a murine model of human GI-cancer. Animals were fed either standard or aspirin supplemented diet (75, 150, or 300 mg/day of human equivalent dose) starting at the age of 4 weeks and continued till the end of the study, while mice were exposed to Si-ions (300 MeV/n; 69 keV/μm) at the age of 8 weeks. Serum PGE2 level, GI tumor size (>2mm), number, and cluster (>5 adjoining tumors) were analyzed at 150 days post-exposure. Aspirin led to a significant reduction in PGE2 in a dose-dependent manner but did not reduce Si-induced GI tumorigenesis even at the highest (300 mg/day) dose. In summary, this study suggests that aspirin could reduce high-LET IR-induced pro-inflammatory PGE2 levels, however, lacks the ability to reduce high-LET IR-induced GI tumorigenesis in Apc mice.
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http://dx.doi.org/10.1016/j.lssr.2021.09.001DOI Listing
November 2021

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

J Proteome Res 2021 11 29;20(11):5145-5155. Epub 2021 Sep 29.

Department of Oncology, Georgetown University Medical Center, Washington, D.C. 20057, United States.

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 Cs deposition. In this study, a novel irradiation system, VAriable Dose-rate External Cs 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 (6,6,6-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 Metabolomics Workbench with study IDs ST001790, ST001791, ST001792, ST001793, and ST001806.
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http://dx.doi.org/10.1021/acs.jproteome.1c00638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9109602PMC
November 2021

A high-throughput and highly automated genotoxicity screening assay.

ALTEX 2022 28;39(1):71-81. Epub 2021 Sep 28.

Department of Oncology, and Department of Biochemistry, Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, USA.

The increasing number of compounds under development and chemicals in commerce that require safety assessments pose a serious challenge for regulatory agencies worldwide. In vitro screening using toxicogenomic biomarkers has been proposed as a first-tier screen in chemical assessment and has been endorsed internationally. We previously developed, evaluated, and validated an in vitro transcriptomic biomarker responsive to DNA damage-inducing (DDI) agents, namely TGx-DDI, for genotoxicity testing in human cells and demonstrated the feasibility of using TGx-DDI in a medium-throughput, cell-based genotoxicity testing system by implementing this biomarker with the Nanostring nCounter system. In this current study, we took advantage of Nanostring nCounter Plexset technology to develop a highly auto­mated, multiplexed, and high-throughput genotoxicity testing assay, designated the TGx-DDI Plexset assay, which can increase the screening efficiency eight-fold compared to standard nCounter technology while decreasing the hands-on time. We demonstrate the high-throughput capability of this assay by eliminating concentration determination and RNA extraction steps without compromising the specificity and sensitivity of TGx-DDI. Thus, we propose that this simple, highly automated, multiplexed high-throughput pipeline can be widely used in chemical screening and assessment.
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http://dx.doi.org/10.14573/altex.2102121DOI Listing
January 2022

Radiochemotherapy upregulates expression of checkpoint receptors on circulating T cells.

Int J Radiat Biol 2021 25;97(11):1563-1568. Epub 2021 Aug 25.

Department of Radiation Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin, China.

Purpose: This study assesses changes of circulating leukocyte subpopulations and the expression of checkpoint receptors in T cells in patients undergoing radiochemotherapy.

Materials And Methods: Fifty-seven patients with either esophageal cancer or cervical cancer who received radiochemotherapy were recruited into this study. Serial blood collection was carried out before and during treatments. Leukocyte subpopulations and the level of PD-1 and CTLA-4 in T cells were determined by flow cytometry. The plasma concentrations of 34 human cytokines, chemokines, and growth factors were quantified.

Results: Significant decreases of lymphocyte count and percentage of T cells and B cells were observed during radiochemotherapy. Percentages of PD-1 and CTLA-4 populations in T cells increased after treatments. Proportion of activated T cells showed no significant difference. No significant changes in the plasma concentrations of the 34 humoral mediators except mild decreases of six cytokines.

Conclusion: Elevated expression of PD-1 and CTLA-4 in T cells in patients receiving radiochemotherapy, which suggests that exhaustion-like T-cell dysfunction develops during cancer cytotoxic treatments.
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http://dx.doi.org/10.1080/09553002.2021.1969051DOI Listing
January 2022

Hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at early stages of white-nose syndrome.

Sci Rep 2021 06 2;11(1):11581. Epub 2021 Jun 2.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, 20057, USA.

White-nose syndrome (WNS) is an emergent wildlife fungal disease of cave-dwelling, hibernating bats that has led to unprecedented mortalities throughout North America. A primary factor in WNS-associated bat mortality includes increased arousals from torpor and premature fat depletion during winter months. Details of species and sex-specific changes in lipid metabolism during WNS are poorly understood and may play an important role in the pathophysiology of the disease. Given the likely role of fat metabolism in WNS and the fact that the liver plays a crucial role in fatty acid distribution and lipid storage, we assessed hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at an early stage of infection with the etiological agent, Pseudogymnoascus destructans (Pd). Differences in lipid profiles were detected at the species and sex level in the sham-inoculated treatment, most strikingly in higher hepatic triacylglyceride (TG) levels in E. fuscus females compared to males. Interestingly, several dominant TGs (storage lipids) decreased dramatically after Pd infection in both female M. lucifugus and E. fuscus. Increases in hepatic glycerophospholipid (structural lipid) levels were only observed in M. lucifugus, including two phosphatidylcholines (PC [32:1], PC [42:6]) and one phosphatidylglycerol (PG [34:1]). These results suggest that even at early stages of WNS, changes in hepatic lipid mobilization may occur and be species and sex specific. As pre-hibernation lipid reserves may aid in bat persistence and survival during WNS, these early perturbations to lipid metabolism could have important implications for management responses that aid in pre-hibernation fat storage.
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http://dx.doi.org/10.1038/s41598-021-90828-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172879PMC
June 2021

Small Molecule Responses to Sequential Irradiation with Neutrons and Photons for Biodosimetry Applications: An Initial Assessment.

Radiat Res 2021 11;196(5):468-477

Department of Oncology, Lombardi Comprehensive Cancer Center.

Mass casualty exposure scenarios from an improvised nuclear device are expected to be far more complex than simple photons. Based on the proximity to the explosion and potential shielding, a mixed field of neutrons and photons comprised of up to approximately 30% neutrons of the total dose is anticipated. This presents significant challenges for biodosimetry and for short-term and long-term medical treatment of exposed populations. In this study we employed untargeted metabolomic methods to develop a biosignature in urine and serum from C57BL/6 mice to address radiation quality issues. The signature was developed in males and applied to samples from female mice to identify potential sex differences. Thirteen urinary (primarily amino acids, vitamin products, nucleotides) and 18 serum biomarkers (primarily mitochondrial and fatty acid β oxidation intermediates) were selected and evaluated in samples from day 1 and day 7 postirradiation. Sham-irradiated groups (controls) were compared to an equitoxic dose (3 Gy X-ray equivalent) from X rays (1.2 Gy/min), neutrons (∼1 Gy/h), or neutrons-photons. Results showed a time-dependent increase in the efficiency of the signatures, with serum providing the highest levels of accuracy in distinguishing not only between exposed from non-exposed populations, but also between radiation quality (photon exposures vs. exposures with a neutron component) and in between neutron-photon exposures (5, 15 or 25% of neutrons in the total dose) for evaluating the neutron contribution. A group of metabolites known as acylcarnitines was only responsive in males, indicating the potential for different mechanisms of action in baseline levels and of neutron-photon responses between the two sexes. Our findings highlight the potential of metabolomics in developing biodosimetric methods to evaluate mixed exposures with high sensitivity and specificity.
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http://dx.doi.org/10.1667/RADE-20-00032.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9004252PMC
November 2021

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

Int J Mol Sci 2021 Mar 17;22(6). Epub 2021 Mar 17.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.

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, O, and Fe) 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.
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http://dx.doi.org/10.3390/ijms22063070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002539PMC
March 2021

Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Arch Toxicol 2021 05 26;95(5):1631-1645. Epub 2021 Mar 26.

Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.
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http://dx.doi.org/10.1007/s00204-021-03014-2DOI Listing
May 2021

3,3'-Diindolylmethane Enhances Tumor Regression After Radiation Through Protecting Normal Cells to Modulate Antitumor Immunity.

Adv Radiat Oncol 2021 Jan-Feb;6(1):100601. Epub 2020 Oct 24.

Department of Oncology, Georgetown University Medical Center, Washington, DC.

Purpose: Preclinical and clinical data indicate that radiation therapy acts as an immune modifier, having both immune-stimulatory and immunosuppressive effects on the tumor-immune microenvironment (TIME). 3.3'-diindolylmethane (DIM) sensitizes tumor cells to radiation and protects mice from lethal doses of total body irradiation. We hypothesize that protecting nontumoral cells from the adverse effects of radiation treatment (RT) may help to correct immunosuppression resulting from radiation.

Methods And Materials: We generated tumor graft models using immune-competent and immune-deficient mouse strains. Narrow-beamed radiation was targeted to tumor sites using shielding. Tumor regression was monitored after DIM and RT versus RT alone. The effects of DIM on the efficacy of RT were assessed using immunohistochemistry staining and gene expression profiling. Complete blood counts, clonogenic cell survival assays, and global gene expression profiling of cultured cells were performed to study DIM's radioprotective effects on normal cells.

Results: DIM enhanced tumor regression after RT in immune-competent but not immune-deficient mice. Data indicated that DIM increased intratumoral immune cells after RT, contributing to enhanced immunologic responses such as adhesion and antigen processing. DIM protected normal cells from radiation-induced immediate injuries in vitro and in vivo Transcriptomic profiling of cultured cells showed that DIM treatment mildly increased expression of some genes that are normally induced after radiation, such as genes involved in cell cycle arrest and apoptosis.

Conclusions: In this study, using cultured cells and preclinical breast cancer models, we show that DIM protects normal cells from radiation-induced immediate cellular injury and combination treatment of DIM and radiation potentiates antitumor immune responses and enhances the efficacy of RT.
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http://dx.doi.org/10.1016/j.adro.2020.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811120PMC
October 2020

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

Sci Rep 2020 11 16;10(1):19899. Epub 2020 Nov 16.

Center for Radiological Research, Columbia University, New York, NY, 10032, USA.

In the long term, Cs 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 Cs, the individual will be exposed to a low dose rate, uniform, whole body, irradiation. The VADER (VAriable Dose-rate External Cs irradiatoR) allows modeling these exposures, bypassing many of the problems inherent in internal emitter studies. Making use of discarded Cs 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 Cs 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 Cs biokinetics in mouse or man. We present the VADER design and characterization of its performance over 18 months of use.
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http://dx.doi.org/10.1038/s41598-020-76941-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670416PMC
November 2020

Serum Metabolomic Alterations Associated with Cesium-137 Internal Emitter Delivered in Various Dose Rates.

Metabolites 2020 Jun 30;10(7). Epub 2020 Jun 30.

Department of Oncology, Georgetown University Medical Center, Washington, DC 20007, USA.

Our laboratory and others have use radiation metabolomics to assess responses in order to develop biomarkers reflecting exposure and level of injury. To expand the types of exposure and compare to previously published results, metabolomic analysis has been carried out using serum samples from mice exposed to Cs internal emitters. Animals were injected intraperitoneally with CsCl solutions of varying radioactivity, and the absorbed doses were calculated. To determine the dose rate effect, serum samples were collected at 2, 3, 5, 7, and 14 days after injection. Based on the time for each group receiving the cumulative dose of 4 Gy, the dose rate for each group was determined. The dose rates analyzed were 0.16 Gy/day (low), 0.69 Gy/day (medium), and 1.25 Gy/day (high). The results indicated that at a cumulative dose of 4 Gy, the low dose rate group had the least number of statistically significantly differential spectral features. Some identified metabolites showed common changes for different dose rates. For example, significantly altered levels of oleamide and sphingosine 1-phosphate were seen in all three groups. On the other hand, the intensity of three amino acids, Isoleucine, Phenylalanine and Arginine, significantly decreased only in the medium dose rate group. These findings have the potential to be used in assessing the exposure and the biological effects of internal emitters.
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http://dx.doi.org/10.3390/metabo10070270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407308PMC
June 2020

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

Metabolites 2020 Jun 8;10(6). Epub 2020 Jun 8.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.

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.
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http://dx.doi.org/10.3390/metabo10060234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345090PMC
June 2020

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

Health Phys 2020 12;119(6):746-757

Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

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.
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http://dx.doi.org/10.1097/HP.0000000000001257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8579862PMC
December 2020

Protons Show Greater Relative Biological Effectiveness for Mammary Tumorigenesis with Higher ERα- and HER2-Positive Tumors Relative to γ-rays in APC Mice.

Int J Radiat Oncol Biol Phys 2020 05 6;107(1):202-211. Epub 2020 Feb 6.

Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC; Department Biochemistry and Molecular & Cellular Biology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC. Electronic address:

Purpose: Exposure to ionizing radiation increases risk of breast cancer. Although proton radiation is encountered in outer space and in medicine, we do not fully understand breast cancer risks from protons owing to limited in vivo data. The purpose of this study was to comparatively assess the effects of γ-rays and protons on mammary tumorigenesis in APC mice.

Methods And Materials: Female APC mice were exposed to 1 GeV protons (1.88 or 4.71 Gy) and Cs γ-rays (2 or 5 Gy). Mice were euthanized 100 to 110 days after irradiation, at which point mammary tumors were scored, tumor grades were assessed, and relative biological effectiveness was calculated. Molecular phenotypes were determined by assessing estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2) status. ERα downstream signaling was assessed by immunohistochemistry.

Results: Exposure to proton radiation led to increased mammary tumor frequency at both proton radiation doses compared with γ-rays. The calculated relative biological effectiveness for proton radiation-induced mammary tumorigenesis was 3.11 for all tumors and >5 for malignant tumors relative to γ-rays. Tumor frequency per unit of radiation was higher at the lower dose, suggesting a saturation effect at the higher dose. Protons induced more adenocarcinomas relative to γ-rays, and proton-induced tumors show greater ERα and HER2 positivity and higher activation of the ERα downstream PI3K/Akt and cyclin D1 pathways relative to γ-rays.

Conclusions: Our data demonstrate that protons pose a higher risk of mammary tumorigenesis relative to γ-rays. We also show that proton radiation-induced tumors in APC mice are ERα- and HER2-positive, which is consistent with our previous data on radiation-induced estrogenic response in wild-type mice. Although this study establishes APC as a model with adequate signal-to-noise ratio for space radiation-induced mammary tumorigenesis, further studies will be required to address the uncertainties in space radiation-induced breast cancer risk estimation.
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http://dx.doi.org/10.1016/j.ijrobp.2020.01.031DOI Listing
May 2020

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

J Am Soc Mass Spectrom 2020 Mar 28;31(3):498-507. Epub 2020 Jan 28.

Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, United States.

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.
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http://dx.doi.org/10.1021/jasms.9b00076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489307PMC
March 2020

Arsenite and cadmium promote the development of mammary tumors.

Carcinogenesis 2020 07;41(7):1005-1014

Department of Oncology, Georgetown University, Washington DC, USA.

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.
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http://dx.doi.org/10.1093/carcin/bgz176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359772PMC
July 2020

Application of the adverse outcome pathway framework to genotoxic modes of action.

Environ Mol Mutagen 2020 01 31;61(1):114-134. Epub 2019 Oct 31.

Pfizer Inc, World Wide Research and Development, Groton, Connecticut.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22339DOI Listing
January 2020

TGx-DDI, a Transcriptomic Biomarker for Genotoxicity Hazard Assessment of Pharmaceuticals and Environmental Chemicals.

Front Big Data 2019 8;2:36. Epub 2019 Oct 8.

Department of Oncology, Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, United States.

Genotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world-wide for analysis of drug candidates, and environmental and industrial chemicals. Current genotoxicity testing batteries feature a high incidence of irrelevant positive findings-particularly for chromosomal damage (CD) assays. The risk management of compounds with positive findings is a major challenge and requires complex, time consuming, and costly follow-up strategies including animal testing. Thus, regulators are urgently in need of new testing approaches to meet legislated mandates. Using machine learning, we identified a set of transcripts that responds predictably to DNA-damage in human cells that we refer to as the TGx-DDI biomarker, which was originally referred to as TGx-28.65. We proposed to use this biomarker in conjunction with current genotoxicity testing batteries to differentiate compounds with irrelevant "false" positive findings in the CD assays from true DNA damaging agents (i.e., for de-risking agents that are clastogenic but not ). We validated the performance of the TGx-DDI biomarker to identify true DNA damaging agents, assessed intra- and inter- laboratory reproducibility, and cross-platform performance. Recently, to augment the application of this biomarker, we developed a high-throughput cell-based genotoxicity testing system using the NanoString nCounter® technology. Here, we review the status of TGx-DDI development, its integration in the genotoxicity testing paradigm, and progress to date in its qualification at the US Food and Drug Administration (FDA) as a drug development tool. If successfully validated and implemented, the TGx-DDI biomarker assay is expected to significantly augment the current strategy for the assessment of genotoxic hazards for drugs and chemicals.
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http://dx.doi.org/10.3389/fdata.2019.00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931968PMC
October 2019

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

Int J Radiat Oncol Biol Phys 2019 11 25;105(4):843-851. Epub 2019 Jul 25.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC; Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC.

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.
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http://dx.doi.org/10.1016/j.ijrobp.2019.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815709PMC
November 2019

Fractionated and Acute Proton Radiation Show Differential Intestinal Tumorigenesis and DNA Damage and Repair Pathway Response in Apc Mice.

Int J Radiat Oncol Biol Phys 2019 11 2;105(3):525-536. Epub 2019 Jul 2.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC; Department of Oncology, Georgetown University Medical Center, Washington, DC; Department of Pathology, Georgetown University Medical Center, Washington, DC. Electronic address:

Purpose: Proton radiation is a major component of the radiation field in outer space and is used clinically in radiation therapy of resistant cancers. Although epidemiologic studies in atom bomb survivors and radiologic workers have established radiation as a risk factor for colorectal cancer (CRC), we have yet to determine the risk of CRC posed by proton radiation owing to a lack of sufficient human or animal data. The purpose of the current study was to quantitatively and qualitatively characterize differential effects of acute and fractionated high-energy protons on colorectal carcinogenesis.

Methods And Materials: We used Apc mice, a well-studied CRC model, to examine acute versus fractionated proton radiation-induced differences in intestinal tumorigenesis and associated signaling pathways. Mice were exposed to 1.88 Gy of proton radiation delivered in a single fraction or in 4 equal daily fractions (0.47 Gy × 4). Intestinal tumor number and grade were scored 100 to 110 days after irradiation, and tumor and tumor-adjacent normal tissues were harvested to assess proliferative β-catenin/Akt pathways and DNA damage response and repair pathways relevant to colorectal carcinogenesis.

Results: Significantly higher intestinal tumor number and grade, along with decreased differentiation, were observed after acute radiation relative to fractionated radiation. Acute protons induced upregulation of β-catenin and Akt pathways with increased proliferative marker phospho-histone H3. Increased DNA damage along with decreased DNA repair factors involved in mismatch repair and nonhomologous end joining were also observed after exposure to acute protons.

Conclusions: We show increased γH2AX, 53BP1, and 8-oxo-dG, suggesting that increased ongoing DNA damage along with decreased DNA repair factors and increased proliferative responses could be triggering a higher number of intestinal tumors after acute relative to fractionated proton exposures in Apc mice. Taken together, our data suggest greater carcinogenic potential of acute relative to fractionated proton radiation.
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http://dx.doi.org/10.1016/j.ijrobp.2019.06.2532DOI Listing
November 2019

Intestinal stem cells acquire premature senescence and senescence associated secretory phenotype concurrent with persistent DNA damage after heavy ion radiation in mice.

Aging (Albany NY) 2019 06;11(12):4145-4158

Department of Biochemistry and Molecular & Cellular Biology and Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.

Heavy ion radiation, prevalent in outer space and relevant for radiotherapy, is densely ionizing and poses risk to stem cells that are key to intestinal homeostasis. Currently, the molecular spectrum of heavy ion radiation-induced perturbations in intestinal stem cells (ISCs), that could trigger intestinal pathologies, remains largely unexplored. The Lgr5-EGFP-IRES-creERT mice were exposed to 50 cGy of iron radiation. Mice were euthanized 60 d after exposure and ISCs were sorted using fluorescence activated cell sorting. Reactive oxygen species (ROS) and mitochondrial superoxide were measured using fluorescent probes. Since DNA damage is linked to senescence and senescent cells acquire senescence-associated secretory phenotype (SASP), we stained ISCs for both senescence markers p16, p21, and p19 as well as SASP markers IL6, IL8, and VEGF. Due to potential positive effects of SASP on proliferation, we also stained for PCNA. Data show increased ROS and ongoing DNA damage, by staining for γH2AX, and 53BP1, along with accumulation of senescence markers. Results also showed increased SASP markers in senescent cells. Collectively, our data suggest that heavy-ion-induced chronic stress and ongoing DNA damage is promoting SASP in a fraction of the ISCs, which has implications for gastrointestinal function, inflammation, and carcinogenesis in astronauts and patients.
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http://dx.doi.org/10.18632/aging.102043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629005PMC
June 2019

Temporal Effects on Radiation Responses in Nonhuman Primates: Identification of Biofluid Small Molecule Signatures by Gas Chromatography⁻Mass Spectrometry Metabolomics.

Metabolites 2019 May 15;9(5). Epub 2019 May 15.

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.

Whole body exposure to ionizing radiation damages tissues leading to physical symptoms which contribute to acute radiation syndrome. Radiation biodosimetry aims to determine characteristic early biomarkers indicative of radiation exposure and is necessary for effective triage after an unanticipated radiological incident. Radiation metabolomics can address this aim by assessing metabolic perturbations following exposure. Gas chromatography-mass spectrometry (GC-MS) is a standardized platform ideal for compound identification. We performed GC time-of-flight MS for the global profiling of nonhuman primate urine and serum samples up to 60 d after a single 4 Gy γ-ray total body exposure. Multivariate statistical analysis showed higher group separation in urine vs. serum. We identified biofluid markers involved in amino acid, lipid, purine, and serotonin metabolism, some of which may indicate host microbiome dysbiosis. Sex differences were observed for amino acid fold changes in serum samples. Additionally, we explored mitochondrial dysfunction by tricarboxylic acid intermediate analysis in the first week with a GC tandem quadrupole MS platform. By adding this temporal component to our previous work exploring dose effects at 7 d, we observed the highest fold changes occurring at 3 d, returning closer to basal levels by 7 d. These results emphasize the utility of both MS-based metabolomics for biodosimetry and complementary analytical platforms for increased metabolome coverage.
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http://dx.doi.org/10.3390/metabo9050098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571779PMC
May 2019

Fabric Phase Sorptive Extraction-A Metabolomic Preprocessing Approach for Ionizing Radiation Exposure Assessment.

J Proteome Res 2019 08 22;18(8):3020-3031. Epub 2019 May 22.

Center for Applied NanoBiosience and Medicine , University of Arizona , 475 North Fifth Street , Phoenix , Arizona 85004 , United States.

The modern application of mass spectrometry-based metabolomics to the field of radiation assessment and biodosimetry has allowed for the development of prompt biomarker screenings for radiation exposure. Our previous work on radiation assessment, in easily accessible biofluids (such as urine, blood, saliva), has revealed unique metabolic perturbations in response to radiation quality, dose, and dose rate. Nevertheless, the employment of swift injury assessment in the case of a radiological disaster still remains a challenge as current sample processing can be time consuming and cause sample degradation. To address these concerns, we report a metabolomics workflow using a mass spectrometry-compatible fabric phase sorptive extraction (FPSE) technique. FPSE employs a matrix coated with sol-gel poly(caprolactone--dimethylsiloxane--caprolactone) that binds both polar and nonpolar metabolites in whole blood, eliminating serum processing steps. We confirm that the FPSE preparation technique combined with liquid chromatography-mass spectrometry can distinguish radiation exposure markers such as taurine, carnitine, arachidonic acid, α-linolenic acid, and oleic acid found 24 h after 8 Gy irradiation. We also note the effect of different membrane fibers on both metabolite extraction efficiency and the temporal stabilization of metabolites in whole blood at room temperature. These findings suggest that the FPSE approach could work in future technology to triage irradiated individuals accurately, via biomarker screening, by providing a novel method to stabilize biofluids between collection and sample analysis.
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http://dx.doi.org/10.1021/acs.jproteome.9b00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437658PMC
August 2019

Author Correction: Lipidomic Signatures of Nonhuman Primates with Radiation-Induced Hematopoietic Syndrome.

Sci Rep 2019 May 3;9(1):6878. Epub 2019 May 3.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, D.C., 20057, USA.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-42491-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499825PMC
May 2019

Impact of inflammatory signaling on radiation biodosimetry: mouse model of inflammatory bowel disease.

BMC Genomics 2019 May 2;20(1):329. Epub 2019 May 2.

Center for Radiological Research, Columbia University Irving Medical Center, New York, NY, 10032, USA.

Background: Ionizing Radiation (IR) is a known pro-inflammatory agent and in the process of development of biomarkers for radiation biodosimetry, a chronic inflammatory disease condition could act as a confounding factor. Hence, it is important to develop radiation signatures that can distinguish between IR-induced inflammatory responses and pre-existing disease. In this study, we compared the gene expression response of a genetically modified mouse model of inflammatory bowel disease (Il10) with that of a normal wild-type mouse to potentially develop transcriptomics-based biodosimetry markers that can predict radiation exposure in individuals regardless of pre-existing inflammatory condition.

Results: Wild-type (WT) and Il10 mice were exposed to whole body irradiation of 7 Gy X-rays. Gene expression responses were studied using high throughput whole genome microarrays in peripheral blood 24 h post-irradiation. Analysis resulted in identification of 1962 and 1844 genes differentially expressed (p < 0.001, FDR < 10%) after radiation exposure in Il10 and WT mice respectively. A set of 155 genes was also identified as differentially expressed between WT and Il10 mice at the baseline pre-irradiation level. Gene ontology analysis revealed that the 155 baseline differentially expressed genes were mainly involved in inflammatory response, glutathione metabolism and collagen deposition. Analysis of radiation responsive genes revealed that innate immune response and p53 signaling processes were strongly associated with up-regulated genes, whereas B-cell development process was found to be significant amongst downregulated genes in the two genotypes. However, specific immune response pathways like MHC based antigen presentation, interferon signaling and hepatic fibrosis were associated with radiation responsive genes in Il10 mice but not WT mice. Further analysis using the IPA prediction tool revealed significant differences in the predicted activation status of T-cell mediated signaling as well as regulators of inflammation between WT and Il10 after irradiation.

Conclusions: Using a mouse model we established that an inflammatory disease condition could affect the expression of many radiation responsive genes. Nevertheless, we identified a panel of genes that, regardless of disease condition, could predict radiation exposure. Our results highlight the need for consideration of pre-existing conditions in the population in the process of development of reliable biodosimetry markers.
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http://dx.doi.org/10.1186/s12864-019-5689-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498469PMC
May 2019
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