Paper
TOCOL PROPHYLAXIS FOR TOTAL-BODY IRRADIATION: A PROTEOMIC
ANALYSIS IN MURINE MODEL
Elliot Rosen,1 Oluseyi O. Fatanmi,2,3 Stephen Y. Wise,2,3 V. Ashutosh Rao,1 and Vijay K. Singh2,3
Abstract—The aim of this study was to analyze the changes in
Downloaded from http://journals.lww.com/health-physics by BhDMf5ePHKbH4TTImqenVAPwFBsBoeDVspUzcFtbCS+CB8iNvRDmLlzF+v7wXXDn on 06/17/2020
mouse jejunum protein expression in response to prophylactic
administration of two promising tocols, g-tocotrienol (GT3) and
a-tocopherol succinate (TS), as radiation countermeasures before
irradiation to elucidate the molecular mechanism(s) of their ra-
dioprotective efficacy. Mice were administered GT3 or TS
(200 mg kg−1) subcutaneously 24 h prior to exposure to 11 Gy
60
Co g-radiation, a supralethal dose for mice. Jejunum was
harvested 24 h post-irradiation. Results of the two-dimensional
differential in-gel electrophoresis (2D-DIGE), coupled with mass
spectrometry, and advanced bioinformatics tools suggest that
the tocols have a corresponding impact on expression of 13 proteins
as identified by mass spectrometry. Ingenuity Pathway Analysis
(IPA) reveals a network of associated proteins involved in inflam-
matory response, organismal injury and abnormalities, and cellu-
lar development. Relevant signaling pathways including actin
cytoskeleton signaling, RhoA signaling, and Rho family GTPase
were identified. This study reveals the major proteins, pathways,
and networks involved in preventing the radiation-induced injury
in gut that may be contributing to enhanced survival.
Health Phys. 119(1):12–20; 2020
Key words: 60Co; gamma radiation; mice; radiation protection
1
Division of Biotechnology Research and Review III, Office of Biotech-
nology Products, Center for Drug Evaluation and Research, U.S. Food and
Drug Administration, Silver Spring, MD; 2Division of Radioprotectants, De-
partment of Pharmacology and Molecular Therapeutics, F. Edward Hébert
School of Medicine, Uniformed Services University of the Health Sci-
ences, Bethesda, MD; 3Armed Forces Radiobiology Research Institute,
Uniformed Services University of the Health Sciences, Bethesda, MD.
The authors declare no conflicts of interest.
For correspondence contact: Vijay K. Singh, Division of Radioprotectants,
Department of Pharmacology and Molecular Therapeutics, F. Edward
Hébert School of Medicine “America's Medical School,” Uniformed
Services University of the Health Sciences, 4301 Jones Bridge Road,
Bethesda, MD 20814, or email at vijay.singh@usuhs.edu.
(Manuscript accepted 14 October 2019)
Supplemental digital content is available in the HTML and PDF
versions of this article on the journal’s website www.health-physics.com.
0017-9078/20/0
Copyright © 2020 The Author(s). Published by Wolters Kluwer
Health, Inc. on behalf of the Health Physics Society. This is an open-access
article distributed under the terms of the Creative Commons Attribution-
Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it
is permissible to download and share the work provided it is properly cited.
The work cannot be changed in any way or used commercially without
permission from the journal.
DOI: 10.1097/HP.0000000000001221
12
INTRODUCTION
NUCLEAR DISASTER, caused by either accidental catastrophe or
deliberate release of radioactive material, would result in a
serious public health challenge and put first responders at
risk of potential exposure. Injury resulting from total- or
partial-body exposure to ionizing radiation manifests as
acute radiation syndrome (ARS) and is characterized by
hematopoietic (2–6 Gy), gastrointestinal (6–8 Gy), or
neurovascular (>8 Gy) sub-syndromes (Hall and Giaccia
2012). Both colony-stimulating factors, granulocyte
colony-stimulating factor (G-CSF/filgrastim and PEGylated
G-CSF/PEGfilgrastim) and granulocyte-macrophage colony-
stimulating factor (GM-CSF/sargramostim) have been ap-
proved by the US Food and Drug Administration (FDA)
and have been procured for the Strategic National Stockpile
(Vendor Managed Inventory) to combat hematopoietic ARS
(H-ARS) in the event of radiological emergency (Farese et al.
2013; Farese and MacVittie 2015; Hankey et al. 2015;
National Institute of Allergic and Infectious Diseases 2015;
US FDA 2015, 2018; Singh and Seed 2018). These agents
provide significant benefit to subjects exposed to radiation;
however, they have also been shown to produce potentially
adverse consequences that should be taken into consider-
ation. For instance, G-CSF administration following treat-
ment with drugs that damage bone marrow stem cells
worsens long-term stem cell damage through disproportion-
ate differentiation stimulation (van Os et al. 2000). Addi-
tional apprehensions arise from G-CSF’s role in exacerbating
delayed lung damage in animal models of ARS (Adachi
et al. 2003; Aeolus Pharmaceuticals 2012). Furthermore,
these agents can only be used as radiomitigators (after radiation
exposure) and not as radioprotectors (prior to exposure).
Therefore, additional agents, particularly radioprotectors,
need to be developed to improve survival for groups like
first responders who are at high risk of radiation exposure.
Finally, these agents must be effective, safe, inexpensive
to produce and store, stable under ambient conditions, and
easily administered.
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 Tocol prophylaxis proteomic analysis c E. ROSEN ET AL.
For the development of such agents to combat radio-
logical or nuclear threats, human efficacy studies are neither
ethical nor feasible. Approval of such drugs will be based on
the FDA Animal Rule (US FDA 2015, 2016). In addition,
extrapolation of the human effective doses for such drugs
needs to be based on animal models according to the Ani-
mal Rule (US FDA 2015, 2016).
Vitamin E analogs, collectively known as tocols (for
structures see Singh et al. 2013), are antioxidant molecules
currently under investigation for their radioprotective po-
tential (Singh et al. 2013, 2016). Both in vitro and in vivo
studies have demonstrated that tocols can reduce the acute
effects of radiation on radiosensitive tissues within the he-
matopoietic and gastrointestinal systems, ultimately increasing
overall survival in radiation-exposed victims (Berbee et al.
2009, Ghosh et al. 2009, Singh et al. 2013). These agents
are efficacious as radioprotectors only when administered
intraparenterally (Singh et al. 2013). Mechanism of action
studies indicate that most of the tocols’ conventional antiox-
idant functions reduce radiation-induced free radicals, which
limits the extent of macromolecular damage to DNA, RNA,
and proteins (Singh et al. 2013). Treatment with tocols has
also been shown to increase circulating levels of radioprotec-
tive cytokines like G-CSF and interleukin-6 (Singh et al.
2010, 2014c; Kulkarni et al. 2013). These cytokines lead to
the mobilization of hematopoietic progenitor cells into the pe-
ripheral circulation, which reduces or prevents radiation-
induced neutropenia, thrombocytopenia, and monocytopenia
(Singh et al. 2014b). Although most of the tocols have
demonstrated radioprotective efficacy, g-tocotrienol
(GT3), a-tocopherol succinate (TS), and d-tocotrienol are
more promising radioprotectors compared to others
(Ghosh et al. 2009; Li et al. 2013; Singh et al. 2014a). All
three agents have dose reduction factors of approximately
1.3 (Hasegawa and Landahl 1970; Singh et al 2014a). In
addition to their antioxidant activity, cellular mechanisms may
play a role in their radioprotective efficacy.
To understand the biological pathways and networks
regulating the cellular radiation response and the complete
molecular mechanism(s) underlying GT3 and TS radiopro-
tective properties, this study analyzes the differential protein
expression in jejunum from mice treated with GT3 or TS
and exposed to g-radiation using two-dimensional differen-
tial in-gel electrophoresis (2D-DIGE) coupled with mass
spectrometry and advanced bioinformatics tools.
MATERIALS AND METHODS
Animals and animal care
Harlan Laboratories, Inc. (Indianapolis, IN) supplied
the male, 6- to 8-wk-old CD2F1 mice. Mice were kept in
quarantine for 1 wk, and microbiological examination con-
firmed the absence of Pseudomonas aeruginosa. Mice were
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13
housed in an Association for Assessment and Accreditation
of Laboratory Animal Care-International accredited facility,
eight per cage, in rooms maintained at 21±2 °C, relative hu-
midity of 50±10%, with a 12 h light/dark cycle and 10–15
cycles of fresh air per hour. Certified rodent rations (Teklad
Rodent Diet, Harlan Laboratories, Inc.) and acidified water
(HCl, pH = 2.5–2.8) were provided to the mice ad libitum.
The study was performed according to the Guide for the
Care and Use of Laboratory Animals of the Institute of Lab-
oratory Animal Resources, National Research Council, US
National Academy of Sciences (National Research Council
of the National Academy of Sciences 2011). All animal pro-
cedures were completed according to a protocol approved
by the Institutional Animal Care and Use Committee.
Experimental groups
Two different mouse experiments were conducted: one
with GT3 and the other with TS. In each experiment, there
were three groups; untreated and sham irradiated, vehicle-
treated and irradiated, and tocol-treated (GT3 or TS) and ir-
radiated. There were eight mice in each treatment group.
Irradiation of mice
Irradiation boxes consisted of compartmentalized Plexi-
glas boxes, designed to hold eight mice. Mice were exposed
to a bilateral, midline dose of 11 Gy 60Co g-radiation at a
dose rate of 0.6 Gy min−1. Radiation dosimetry was estab-
lished primarily on the alanine/EPR (electron paramagnetic
resonance) system, currently accepted as one of the most ac-
curate methods for relatively high radiation doses and used
for intercomparison between national metrology institutions.
The calibration curves (spectrometer e-Scan, Bruker Biospin,
Inc., Madison, WI) used in dose measurements are based on
standard alanine calibration sets procured from the US Na-
tional Institute of Standards and Technology (NIST, Gaithers-
burg, MD) as described earlier (Nagy 2000; Singh et al.
2013). The alanine dosimeters obtained from NIST had been
calibrated in terms of absorbed dose to water using the US
national standard radiation sources. Identical alanine dosim-
eters were placed midline within mouse phantoms (Plexiglas
2.5 cm in diameter, 7.5 cm length) and irradiated for predefined
periods of time. Measurement of their EPR signals using the
calibration curve were constructed with alanine dosimeters from
NIST-provided dose rates to water in the core bodies of
mice. A small correction was subsequently applied for the
difference in mass energy absorption coefficients between
water and soft tissue.
Drug preparation and administration
Yasoo Health, Inc. (Johnson City, TN) supplied GT3
formulation in 5% Tween-80 in saline. TS was purchased
from Sigma-Aldrich (St. Louis, MO) and suspended in
polyethylene glycol (PEG)-400 and Tween-80. GT3 and
TS formulations were adjusted to a final concentration of
200 mg kg−1 in 0.1 mL total volume. Equal volumes of olive
14 Health Physics July 2020, Volume 119, Number 1

Fig. 1. Differential protein expression in mouse jejuna following prophylactic vitamin E treatment before radiation exposure. (A) Two-dimensional
difference in gel electrophoresis (2D-DIGE) of jejuna tissue lysate from nontreated (red) and TS-treated (green) animals receiving TBI. Samples are
labeled with Cy5 (red) or Cy3 (green) fluorescent dye; internal control (pooled sample) is labeled with Cy2 (yellow). (B) Three-dimensional
densiometric visualization of gel spot IDs 87 and 74, corresponding to peroxiredoxin-1 and phosphoglycerate mutase 1. Left: tocol succinate-
treated, Right: nontreated.

oil were administered as the vehicle (olive oil in 5% Tween-80
in saline for GT3 or PEG-400 and Tween-80 for TS). A
Luer-Lock syringe with a 23 G needle was used for all drug
administrations. Subcutaneous injections were administered
at the nape of the neck at 24 h before irradiation.
Tissue collection and sample preparation
At 24 h post-radiation exposure, mice were anes-
thetized using isoflurane and humanely euthanized by
exsanguination. The jejunum sections (about 5 cm long)
were collected and flushed with phosphate-buffered saline
(PBS), flash frozen on dry ice (−78.5 °C), and stored at
−80 °C. Tissue samples were homogenized in 2D lysis
buffer consisting of 30 mM Tris-HCl (pH 8.8), 7 M urea,
2 M thiourea, and 4% CHAPS (3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate) detergent. Final
concentrations were adjusted to 4–8 mg mL−1 of protein

Fig. 2. Table of correspondingly up/down-regulated proteins following treatment with isoforms of vitamin E before radiation exposure. (A) Heat
map displaying relative upregulation (red) or downregulation (green) of jejuna proteins expressed as ratio of irradiated to irradiated and drug treated
mice. (B) Average protein expression fold change ratio of irradiated to unirradiated and irradiated to irradiated and drug-treated mice. GT3 (n = 6)
expression ratio is followed by TS (n = 3) in each column. Selection criteria for irradiated to irradiated and drug-treated mice ratio included average
fold change of 1.5 or greater and p<0.05. NT = no treatment, ND = not detected.
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 Tocol prophylaxis proteomic analysis c E. ROSEN ET AL. 15

for analysis. The protein lysate was quantified using the
bicinchoninic acid assay. Samples were frozen at −80 °C
until analysis.
Protein identification
Jejunal lysates were labeled with two different Cy dyes
(Cy3 or Cy5). As an internal control, lysates of eight mice
from vehicle control and tocol-treated groups were pooled
and labeled with Cy2. Samples were separated by 2D-DIGE,
and all 2D Gels were run in triplicate as described earlier
(Kongara et al. 2010). The 2D gels were then analyzed using
an Amersham Biosciences 2D-gel system (Amersham Bio-
sciences, Piscataway, NJ). Images were scanned using Ty-
phoon TRIO and analyzed by ImageQuantTL software (GE
Healthcare, Chicago, IL). In-gel and cross-gel analysis em-
ployed Decyder software version 6.5 (GE Healthcare). Be-
tween 150 and 200 protein spots per gel were selected for
identification using Ettan Spot picker (Amersham Biosciences).
The selected spots were then digested in buffer contain-
ing sequencing grade modified trypsin protease at 37 °C.
The digested peptides were desalted and incorporated into
a-cyano-4-hydroxy-cinnamic acid matrix and spotted on a
MALDI (matrix assisted laser desorption/ioniziation) plate.
MALDI time of flight tandem mass spectrometry (MALDI-
TOF/TOF) was performed using an Applied Biosystems
Proteomic Analyzer Spectrometer. Protein identification
was based on peptide fingerprinting mass mapping and pep-
tide fragmentation mapping. Peptide mass and associated
fragmentation spectra were submitted to a GPS Explorer
work station equipped with MASCOT search engine (Matrix
Science, Boston, MA) to identify proteins from a primary se-
quence database.
Ingenuity Pathway Analysis (IPA) software (Qiagen,
Redwood City, CA) was then used to integrate the proteins
into pathways and networks based on biological and/or
functional relevance to the other proteins as well as the sig-
nificance value of uploaded proteins. The groups of proteins
were first considered for each individual drug treatment be-
fore common proteins of interest were identified. Networks
and signaling pathways were informed by searching for re-
lationships based on the specific tissue type being consid-
ered, mouse small intestine.
Statistical analysis
Statistical significance was assessed by Student’s t-test
analysis. Significant change of protein abundance was defined

Fig. 3. IPA reveals network of common proteins involved in inflammatory response, organismal injury and abnormalities, and cellular develop-
ment. Proteomic analysis highlighting network of related proteins found to be simultaneously up- or down-regulated in response to vitamin E
isoform treatment. Relative upregulation (red) and downregulation (green) ratio of irradiated to irradiated and drug-treated mice.
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16 Health Physics July 2020, Volume 119, Number 1

as at least 1.5-fold difference with a p value < 0.05 (n = 3). For
MALDI-TOF/TOF analysis, only those proteins with a protein
score confidence interval percentage or ion confidence in-
terval percentage of >95% were considered significant.
RESULTS
The aim of this study was to analyze the changes in the
protein expression profiles for two promising radioprotectants
under development, GT3 and TS, in irradiated mice to better
understand the molecular mechanism of their radioprotective
efficacy. Mice were treated with GT3, TS or respective vehicle
24 h prior to supralethal irradiation (11 Gy, which causes
gastrointestinal injury in addition to hematopoietic sub-
syndrome). The jejunum was collected 24 h post-irradiation,
and the fold-change protein expression profile was determined
by 2D-DIGE. The spot detection and investigation of protein
abundance in each gel provided ~2,500 proteins of interest.
From each gel, about 100–200 spots with isoelectric points
ranging between pI 4 and 9 and molecular weights ranging
from 10 to 150 kDa were digested, separated, and identified
using MALDI-TOF/TOF and the MASCOT search engine
(Fig. 1). From the studies performed independently with
GT3 and TS, the names of common proteins showing a
statistically significant (p<0.05) fold difference of 1.5 or
greater with ratio of total-body irradiation (TBI) to unirradi-
ated mice are presented (please see Supplemental Digital
Content 2, Table 1, http://links.lww.com/HP/A178). Of this
group of 26 total proteins reflecting a change with irradia-
tion, tocol prophylaxis was found to maintain the levels of
13 proteins with respect to unirradiated mice (Fig. 2). Basal
expression of cytochrome b5 was not detected in one of the
two studies, denoted as not detected (ND) in the figure. The
group of proteins showing maintained expression levels
with tocol treatment include proteins involved in the struc-
ture and remodeling of cytoskeleton, protein synthesis, ca-
talysis of reduction of hydrogen peroxide and organic
hydroperoxides, regulation of apoptosis, modulation of cell-
cell and cell-matrix interactions, and endosomal sorting.
Evaluating these proteins as a group with IPA revealed
a network of common proteins involved in cell cycle, gene
expression, and associated with cardiovascular disease
(Fig. 3). The proteomic analysis suggests a network of re-
lated proteins simultaneously up- or down-regulated in re-
sponse to prophylactic vitamin E isoform treatment given
prior to 11 Gy irradiation, maintaining the levels associated
with unirradiated animals. Further investigation of this group
of proteins showing a change in expression in response to

Fig. 4. Actin cytoskeleton signaling. IPA reveals signaling relationships shared by vitamin E isoform treatments. Relative upregulation (red) and
downregulation (green) ratio of irradiated to irradiated and drug-treated mice protein expression observed in the two drug studies. Proteins without
color are predicted to be influenced based on signaling relationships in small intestine of mouse.
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 Tocol prophylaxis proteomic analysis c E. ROSEN ET AL. 17

Fig. 5. RhoA signaling. IPA reveals signaling relationships shared by vitamin E isoform treatments. Relative upregulation (red) and downregula-
tion (green) ratio of irradiated to irradiated and drug-treated mice protein expression observed in the two drug studies. Proteins without color are
predicted to be influenced based on signaling relationships in the small intestine of the mouse.

irradiation reveals relationships including actin cytoskeleton
signaling (Fig. 4), RhoA signaling (Fig. 5), and Rho family
GTPase signaling (Fig. 6).
We also considered the vitamin E isoform treatments
individually. When jejunal lysate from healthy mice was
compared to that of mice exposed to 11 Gy and prophylac-
tically administered the vehicle, 48 proteins including isomers
of the same proteins were differentially expressed. When jejunal
lysate from healthy mice was compared to that of mice ex-
posed to 11 Gy and prophylactically administered TS, 18
proteins were differentially expressed. Finally, when jejunal
lysate from irradiated mice were compared to that of TS-treated/
irradiated mice, 77 proteins were differentially expressed.
Radiation exposure caused the differential expression
of 48 of the 108 jejunal proteins (irradiated vs. unirradi-
ated). Of these 48 proteins, all could be identified, and
34 had ratios with the same directionality between the
two group comparisons (irradiated vs. unirradiated and ir-
radiated vs. drug-treated/irradiated), indicating the drug's
ability to prevent protein expression alteration by radiation ex-
posure (please see Supplemental Content 2, Table 2, http://
links.lww.com/HP/A178).
IPA analysis of these 34 proteins reflecting the same
directionality yielded the five top canonical pathways:
signaling by Rho family GTPases, glycolysis I, xenobiotic
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metabolism signaling, 14-3-3-mediated signaling, and reti-
nol biosynthesis. The top five diseases and disorders for
which these proteins are involved are inflammatory response,
cancer, organismal injury and abnormalities, gastrointestinal
disease, and hepatic system disease. The molecular and cellu-
lar functions to which these proteins pertain are cell death and
survival, cell morphology, cellular assembly and organiza-
tion, as well as cellular compromise and development (please
see Supplemental Digital Content 2, Table 3, http://links.
lww.com/HP/A178). The top network associated functions
are cellular compromise (cellular stress and immune re-
sponse), cell morphology, and cellular assembly and organi-
zation (please see Supplemental Digital Content 1, Figure 1,
http://links.lww.com/HP/A177) (Singh et al. 2018). The
second top network-associated functions are cancer, neuro-
logical disease, and organismal injury and abnormalities
(please see Supplemental Digital Content 1, Figure 2, http://
links.lww.com/HP/A177).
Considering the GT3 treatment independently, when
jejunal lysate from healthy mice was compared to that of
mice exposed to 11 Gy and prophylactically administered
the vehicle, 142 proteins, including isomers of the same pro-
teins, were differentially expressed. When jejunal lysate
from healthy mice was compared to that of mice exposed
to 11 Gy and prophylactically administered GT3, 21
18 Health Physics July 2020, Volume 119, Number 1

Fig. 6. Rho family GTPases signaling. IPA reveals signaling relationships shared by vitamin E isoform treatments. Relative upregulation (red) and
downregulation (green) ratio of irradiated to irradiated and drug-treated mice protein expression observed in the two drug studies. Proteins without
color are predicted to be influenced based on signaling relationships in small intestine of mouse.

proteins were differentially expressed. When jejunal lysate
from irradiated mice was compared to that of GT3-treated/
irradiated mice, 65 proteins were differentially expressed.
Radiation exposure caused the differential expression
of 142 of the 163 jejunal proteins (irradiated vs. unirradi-
ated). Of these 142 proteins, 112 could be identified and
52 showed ratios with the same directionality between the
two group comparisons (irradiated vs. unirradiated and irra-
diated vs. drug-treated/irradiated), indicating a drug-effect,
preventing protein expression alteration by radiation exposure
(please see Supplemental Digital Content 2, Table 4, http://
links.lww.com/HP/A178). IPA analysis of jejuna proteins
yielded the five top canonical pathways including calcium
signaling, actin cytoskeleton signaling, epithelial adherens
junction signaling, RhoGDI signaling, and agranulocyte
adhesion and diapedesis (please see Supplemental Digital
Content 2, Table 5, http://links.lww.com/HP/A178). The
top five diseases and disorders for which these proteins
are associated are inflammatory response, dermatological
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diseases and conditions, hematological disease, metabolic
disease, and organismal injury and abnormalities. The mo-
lecular and cellular functions include cell death and survival,
cellular development, cellular growth and proliferation,
cell signaling, and post-translational modification. The
top network-associated functions are cell signaling, post-
translational modification, and protein synthesis (please
see Supplemental Digital Content 1, Figure 3, http://links.lww.
com/HP/A177) (Singh et al. 2018). The second top network-
associated functions are inflammatory response, nutritional
disease, and free radical scavenging (please see Supplemental
Digital Content 1, Figure 4, http://links.lww.com/HP/A177).
DISCUSSION
Proteome-wide analysis by 2D-DIGE has broadened
our understanding of cellular responses to damage–causing
agents like ionizing radiation revealing vital cellular pro-
teins, pathways, and networks that operate during the
 Tocol prophylaxis proteomic analysis c E. ROSEN ET AL.
damage response (Stickel et al. 2014). Recent studies sug-
gest a non-conventional mechanism of action through the
release of radioprotective cytokines and growth factors (G-
CSF) that facilitates the immune response and hematopoi-
etic progenitor cell mobilization into peripheral circulation.
The various intracellular and extracellular molecular fac-
tors, signaling pathways, and potential direct and indirect
targets of tocols that contribute to enhanced survival in mice
are still not known. Therefore, the present study examines
the GT3 and TS mediated maintenance of protein expres-
sion levels to examine the cellular responses to radiation
using a high throughput 2D-DIGE approach. The identified
list of proteins was further analyzed using the web-based
software application IPA.
By combining the control groups of the two tocol stud-
ies, we identified 26 common proteins showing a change in
expression due to radiation exposure. Interestingly, 13 of
these changes in protein expression were found to have been
corrected by the tocol prophylaxis, suggesting that the
mechanism(s) of action for both tocols might be the same.
Several proteins identified can be classified as cytoskeletal
proteins or proteins involved with the remodeling and
development of the cytoskeleton: dihydropyrimidinase-
related protein 2, ezrin, elongation factor 2, and plastin-1.
Alternatively, cytoplasmic actin 2 was upregulated with
TBI, and tocol treatment reversed this effect. An impact
on cytoskeletal dynamics appears to be consistent with pre-
vious findings (Gabrys et al. 2007; Cheema et al. 2018). Of
note, antioxidant enzyme peroxiredoxin-1 levels were de-
creased with irradiation, and this change was prevented with
tocol treatment. While peroxiredoxin-1 has been found to
be upregulated in human HT29 colon cancer and rat C6 gli-
oma cells in response to irradiation, perhaps the oncologic
status explains the difference in response in healthy tissue
(Chen et al. 2002). A major regulator of apoptotic pathways,
cell cycle progression, signal transduction, and checkpoint
activation, 14-3-3 protein zeta/delta, was found to be down-
regulated with irradiation, and the change in expression was
prevented with tocol treatment (Brunet et al. 2002). Finally,
vacuolar protein-sorting-associated protein 25 was found to
be downregulated following irradiation, indicating a change
in endosomal sorting activity, which in turn would impact
normal lysosomal degradation (Im et al. 2009). Tocol treat-
ment appeared to prevent this change in activity.
When we turn to consider the group of proteins that
were modified following irradiation and these changes were
prevented by tocol treatment, IPA reveals a network of com-
mon proteins involving inflammatory response, organismal
injury and abnormalities, and cellular development. Further
proteomic analysis identifies three major signaling pathways
impacted by irradiation and attenuated with tocol treatment:
actin cytoskeleton, RhoA, and Rho family GTPases signal-
ing. When the tocol studies are considered individually, we
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19
see similar networks at play. In the TS study, we found mul-
tiple proteins representing a network related to functions in-
cluding cellular compromise, cell morphology, and cellular
assembly and organization. Furthermore, we found a net-
work of associated functions including cancer, neurological
disease, and organismal injury and abnormalities as might
be expected with radiation exposure. Considering the GT3
study, the top network revealed associated functions of cell
signaling, post-translational modification, and protein
synthesis that appears to be consistent with the TS study.
Interestingly, another top network of associated functions
includes inflammatory response and free radical scavenging
in response to TBI.
Recent publications from Byrum et al. (2016, 2017)
note proteomic changes in the plasma and urine proteomes
in response to TBI in a nonhuman primate model. These
studies suggest that the intestinal damage may be detected
rapidly by sampling the plasma and/or urine (Byrum et al.
2016, 2017). Histopathology of jejunum from irradiated
and tocol-treated animals (specifically GT3 and TS) has
been published earlier, and there is definite improvement
in the gut of tocol-treated and irradiated animals compared
with irradiated animals receiving no treatment (Berbee
et al. 2009; Singh et al. 2012). Such improvement may be
related to change in the protein expression. Tocols in com-
bination with radiation exposure can be pro-oxidative (lipid
peroxidation), and such lipid peroxidation may influence
proteomes. Considered as a whole, the present study demon-
strates the inflammation-associated and antioxidant proteomic
changes in response to prophylactic use of GT3 and TS,
warranting further investigation of such prophylactic treatment
toward radioprotection and their mechanism(s) of action.
Acknowledgments—Authors are thankful to Victoria L Newman, Joshua Starr,
and Amit Verma for their help. This work was funded by a grant from the
Armed Forces Radiobiology Research Institute (RAB2HD) to VKS.
Disclosure—The views expressed in this article are those of the authors
and do not necessarily reflect the official policy or position of the US Food
and Drug Administration and the Department of Health and Human Services
or the Uniformed Services University of the Health Sciences and the US De-
partment of Defense. Mention of trade names, commercial products, or organi-
zations does not imply endorsement by the United States Government.
Author contributions—VKS conceived and planned the experiments.
OOF and SYW carried out the experiments. ER performed the IPA analysis.
ER, VAR, and VKS prepared the manuscript. All authors discussed the results
and contributed to the final manuscript.
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