Life Sciences in Space Research 31 (2021) 43–50

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Life Sciences in Space Research

journal homepage: www.elsevier.com/locate/lssr

Mitigation of late cardiovascular effects of oxygen ion radiation by

γ-tocotrienol in a mouse model
Ashley S. Nemec-Bakk a, *, Vijayalakshmi Sridharan a, Reid D. Landes b, Preeti Singh a,
Maohua Cao c, John W. Seawright d, Xingui Liu e, Guangrong Zheng f, Paari Dominic g,
Rupak Pathak a, Marjan Boerma a
a
Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA
b
Department of Biostatistics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
c
College of Dentistry, Texas A&M University, Dallas TX, USA
d
McLennan Community College, Waco, TX, USA
e
Department of Pharmacodynamics, University of Florida, Gainesville, FL, USA
f
Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA
g
Department of Medicine and Center of Excellence for Cardiovascular Diseases & Sciences, Louisiana State University Health Sciences Center, Shreveport, LA, USA

A R T I C L E I N F O A B S T R A C T

Key words: Purpose: While there is concern about degenerative tissue effects of exposure to space radiation during deep-
High-LET radiation space missions, there are no pharmacological countermeasures against these adverse effects. γ-Tocotrienol
Cardiovascular system (GT3) is a natural form of vitamin E that has anti-oxidant properties, modifies cholesterol metabolism, and has
Degenerative tissue effects
anti-inflammatory and endothelial cell protective properties. The purpose of this study was to test whether GT3
γ-Tocotrienol
could mitigate cardiovascular effects of oxygen ion (16O) irradiation in a mouse model. Materials and methods:
Radiation countermeasure
Male C57BL/6 J mice were exposed to whole-body 16O (600 MeV/n) irradiation (0.26–0.33 Gy/min) at doses of
0 or 0.25 Gy at 6 months of age and were followed up to 9 months after irradiation. Animals were administered
GT3 (50 mg/kg/day s.c.) or vehicle, on Monday – Friday starting on day 3 after irradiation for a total of 16
administrations. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters.
Cardiac tissue remodeling and inflammatory infiltration were assessed with histology and immunoblot analysis
at 2 weeks, 3 and 9 months after radiation. Results: GT3 mitigated the effects of 16O radiation on cardiac
function, the expression of a collagen type III peptide, and markers of mast cells, T-cells and monocytes/mac­
rophages in the left ventricle. Conclusions: GT3 may be a potential countermeasure against late degenerative
tissue effects of high-linear energy transfer radiation in the heart.

1. Introduction (LET) radiation exposures of human populations described above,

Studies of Japanese atomic bomb survivors (Preston et al., 2003;
Ozasa et al., 2016) and other human populations exposed to low doses of
ionizing radiation to a large part of the body (Little et al., 2012; Simo­
netto et al., 2014; Carr et al., 2005) have shown an increased rate in
cardiovascular disease. This has raised the concern about potential
degenerative tissue effects in the cardiovascular system due to low-dose
exposure to ionizing radiation during deep space travel (Chancellor
et al., 2014; Hellweg and Baumstark-Khan, 2007; NCRP 2006). Since the
biological properties of the high-Z high-energy (HZE) particle radiation
in deep space are different from those of the low-linear energy transfer
research is needed to identify the risk of cardiovascular disease from
exposure to space radiation.
Currently, there is minimal opportunity to obtain data on biological
effects of HZE particles from human subjects, which makes the deter­
mination of the biological response almost exclusively dependent on
animal models (Sridharan et al., 2020; Garikipati et al., 2021; Sasi et al.,
2017; S.S. Ramadan et al., 2016; Koturbash et al., 2016; Miousse et al.,
2019). Studies in rodent models of HZE radiation exposure have shown
alterations in cardiac function and cardiac tissue remodeling (X. Yan
et al., 2014; X. Yan et al., 2014), indicators of inflammation in the heart
(Tungjai et al., 2013) and vascular stiffness and endothelial dysfunction

* Corresponding author at: 4301 West Markham Slot 522-10, Little Rock, AR 72205, USA.
E-mail address: anemecbakk@uams.edu (A.S. Nemec-Bakk).

https://doi.org/10.1016/j.lssr.2021.07.006
Received 13 May 2021; Received in revised form 2 July 2021; Accepted 29 July 2021
Available online 3 August 2021
2214-5524/© 2021 The Committee on Space Research (COSPAR). Published by Elsevier B.V. All rights reserved.
A.S. Nemec-Bakk et al.
(Soucy et al., 2011).
Since astronauts in deep space will spend most of their time inside a
space craft, it is important that research models for the assessment of
health effects of deep space travel mimic the radiation environment that
may be found inside the space craft as opposed to free space. Frag­
mentation of heavy ions in the wall of a space craft will lead to a radi­
ation environment inside the craft that, for a large part, consists of ions
of Z ≤ 10 (Walker et al., 2013). Therefore, we previously performed a
study in which adult male C57BL/6J mice were exposed to protons (150
MeV) or oxygen ions (16O, 600 MeV/n) (Seawright et al., 2019). We
found that 16O at doses from 0.05 to 1 Gy caused small changes in
cardiac function and induced the expression of left ventricular collagen
type III and left ventricular protein levels of mast cell tryptase, CD2
(marker of T-cells) and CD68 (monocytes/macrophages). In the current
project, we used the same mouse model to test whether a radiation
countermeasure may mitigate these effects of 16O exposure.
γ-Tocotrienol (GT3) is a natural form of vitamin E that has long been
known for its properties as protector against the acute radiation syn­
drome from exposure to low-LET radiation (Berbee et al., 2009; Singh
et al., 2011; Ghosh et al., 2009). While GT3 has anti-oxidant properties
similar to those of the most common form of vitamin E, α-tocopherol
(Yoshida et al., 2003; Suarna et al., 1993), GT3 has additional benefits. It
is a potent inhibitor of 3‑hydroxy-3-methyl-glutaryl-coenzyme A
(HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosyn­
thesis (Parker et al., 1993; Song and Bose-Boyd, 2006). Moreover, GT3
accumulates in endothelial cells to at least 30-fold higher levels than
α-tocopherol and modulates larger numbers of genes in these cells
(Berbee et al., 2012; Naito et al., 2005). We have previously shown that
GT3 exerts its radiation protection at least in part by inhibiting the
cholesterol synthesis pathway (Berbee et al., 2009) and by reducing
endothelial dysfunction (Pathak et al., 2015). Altogether, we hypothe­
size that GT3 has beneficial effects in the cardiovascular response to
ionizing radiation and tested whether administration of GT3 would alter
the effects of a single dose of 16O (600 MeV/n, 0.25 Gy) on cardiac
function and structure in male C57BL/6J mice.
2. Materials and methods
2.1. Animal housing and radiation
All animal procedures within this study were approved by the
Institutional Animal Care and Use Committees of the University of
Arkansas for Medical Sciences (UAMS) and Brookhaven National Lab­
oratory (BNL). Male C57BL/6J mice (n = 140 total; Jackson Laboratory;
Bar Harbor, ME) were housed at UAMS, 5 per cage on a 12:12 hour light
: dark cycle and were administered standard rodent chow low in soy
(2020X, Harlan Laboratories) and water ad libitum. The cages were
randomly divided into 4 experimental groups: 16O or sham-irradiation,
each with vehicle or GT3 administration after irradiation (n = 35 mice
per group).
At 6 months of age, all mice were transported to Brookhaven Na­
tional Laboratory (BNL; Upton, NY) by overnight air and housed on a
12:12 hour light:dark cycle, 2020X diet and water ad libitum. After 1
week acclimatization to BNL, mice were exposed to whole-body 16O
irradiation at the NASA Space Radiation Laboratory (NSRL). Since in a
previous study we had tested the effects of 16O in a dose range of 0.05 –
1 Gy (Seawright et al., 2019), we selected 0.25 Gy as an intermediate
dose for the current study. For this purpose, mice were individually
positioned in a clear Lucite cube, placed within the NSRL beam line and
were exposed to 16O (600 MeV/n) at a dose rate of 0.26 − 0.33 Gy/min.
Radiation dosimetry was performed by the NSRL physics team.
Sham-irradiated mice were also transported to NSRL and placed in the
clear Lucite cubes for about 1 min (the same time as the irradiated mice),
but were not exposed to 16O. Two days after irradiation or
sham-treatment, mice were returned to UAMS. Within each experi­
mental group, 10 mice were scheduled for sacrifice at 2 weeks after
44
Life Sciences in Space Research 31 (2021) 43–50
radiation, 10 mice at the 3 months-time point, and 15 mice at 9 months.
2.2. GT3 administration and plasma measurement
GT3 was separated from DeltaGold® using flash column with silica
gel (230 – 400 mesh) as the stationary phase. Gradient elution was
performed with ethyl acetate and hexanes (20/1 to 10/1). Purity of GT3
(> 95%) was determined with gas chromatography- and liquid
chromatography-mass spectrometry.
Subcutaneous administration of GT3 (50 mg/kg bodyweight once a
day) or vehicle (saline, with 5% Tween 80) started on day 3 after irra­
diation (a Friday) and continued on Monday – Friday for a total of 16
administrations. Plasma samples of mice sacrificed 2 weeks after irra­
diation, as described under the heading Blood and tissue collection, were
used to assess plasma concentration of GT3 using high-pressure liquid
chromatography (HPLC).
2.3. High-resolution ultrasonography
Ultrasonography was performed at 3, 5, 7 and 9 months after irra­
diation (n = 15 mice per group) by one investigator who was blinded to
the treatment groups. The day before ultrasonography, mice were
anesthetized with 2% isoflurane, and hair was removed from the thorax
and abdomen using a depilatory cream. On the day of ultrasonography,
mice were anesthetized with 1.5 – 2% isoflurane and placed supine on a
heated platform that monitors respiration rate and ECG. The mice were
scanned with a Vevo® 2100 imaging system (VisualSonics, Inc.) with a
MS400 (18–38 MHz) transducer. Echocardiographs were obtained in the
short axis M-mode at the mid-left ventricular level.
Pulsed-wave Doppler was used to determine abdominal aorta blood
flow as an indicator of both cardiac and vascular function. For this
purpose, the probe was placed in the transverse axis, immediately
anterior of the renal artery branch point.
The Vevo® LAB cardiac software package was used to obtain ultra­
sonography parameters from three M-mode scans per animal, and the
vascular package was used to assess blood flow parameters from three
consecutive beats per abdominal aorta Pulsed-wave Doppler scan, in up
to three scans per animal. A description of all parameters obtained from
ultrasonography is provided in Supplemental Materials, Table S1.
2.4. Blood and tissue collection
Two weeks, 3 months, and 9 months after irradiation, mice were
weighed in preparation for blood and tissue collection. Calipers were
used to measure tibia length as an additional indicator of body size. Mice
were anesthetized with 3% isoflurane, and a modified infusion set (27 G
with shortened tubing) was used to inject a single dose of heparin
(30− 40 U/kg) into the abdominal vena cava. Without changing the
position of the infusion set, a blood sample was drawn and transferred
into an ethylenediaminetetraacetic acid (EDTA) coated tube. Immedi­
ately after blood collection, the heart was collected, rinsed, weighed and
cut longitudinally. One half of the heart was fixed in 5% formalin for 24
h and embedded in paraffin. For the other half, a specimen of left
ventricle was processed as described under the heading Electron micro­
scopy. The remainder of the heart was divided into atria, right ventricle,
and 4 samples of left ventricle and snap-frozen in liquid nitrogen.
2.5. Blood chemistry values
Blood samples collected at 9 months were used to measure blood
chemistry values with an i-STAT blood analyzer and CHEM8 + car­
tridges (Abbott Labs, Lake Bluff, IL), and blood glucose levels were
determined by using FreeStyle Precision Neo blood glucose monitoring
system and test strips (Abbott Laboratories, Lake Bluff, IL). Values were
determined for blood anion gap (ANGAP), blood concentration of urea
nitrogen (BUN), Cl, blood concentration of creatine (CREA), glucose,
A.S. Nemec-Bakk et al.
hemoglobin (HB), hematocrit (HCT), K, Na, concentration of free
ionized calcium (ICA), and blood concentration of carbon dioxide
(TCO2).
2.6. Histology
For the assessment of cardiac collagen deposition, 5 µm longitudinal
sections of heart were rehydrated and incubated with Sirius Red sup­
plemented with Fast Green. Sections were scanned with a ScanScope
CS2 slide scanner and analyzed with ImageScope 12 software (Leica
Biosystems, Wetzlar, Germany). The relative tissue area of collagens was
calculated as the red-stained area expressed as a percentage of the total
tissue area of each section.
2.7. Immunohistochemistry
Immunohistochemistry was used to identify cluster of differentiation
(CD) 45-positive cells. Left ventricle sections were rehydrated and an­
tigen retrieval was performed by incubating the sections in Target
Retrieval buffer (DAKO, Glostrup, Denmark) at 120 ◦ C in a decloaking
chamber (Biocare Medical, Pacheco, CA) for 20 min, cooling for 45 min
in the retrieval buffer, and then were rinsed in distilled water. Imme­
diately after, sections were then incubated in 1% H2O2 in methanol to
block endogenous peroxidase, followed by 10% normal serum in 3% dry
milk powder and 0.2% bovine serum albumin. Sections were incubated
overnight with rat anti-CD45 (Santa Cruz Biotechnology, Santa Crus,
CA), followed by biotinylated goat anti-rat IgG (Bio-Rad, Hercules, CA)
and an avidin-biotin-peroxidase complex (Vector Laboratories, Burlin­
game, CA) for 45 min. Antibodies were visualized with 0.5 mg/mL 3, 3′ -
diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxy­
lin. Stained sections were examined with an Axioskop transmitted-light
microscope (Carl Zeiss), and CD45-positive cells were counted in 10
optical areas per section. Cells were counted by an individual who was
aware of the animal identification numbers but not the treatment
groups. The number of CD-positive cells were divided by the total area of
the 10 views.
2.8. Electron microscopy
Tissue specimens of left ventricle were fixed and processed for
electron microscopy using a method described by Cocchiaro et al.
(Cocchiaro et al., 2008). Sections were analyzed with a Tecnai F20 200
keV electron microscope (FEI Company, Hillsboro OR).
2.9. Immunoblot analysis
To determine protein expression indicative of cardiac remodeling
and inflammatory infiltration, immunoblot analysis was performed. A
Potter-Elvehjem mechanical compact stirrer (BDC2002, Caframo Lab
Solutions, Georgian Bluffs, Ontario) was used to homogenize left ven­
tricular samples in a 1% Triton-X100 RIPA buffer containing protease
(1:100, Sigma-Aldrich, St Louis, MO) and phosphatase inhibitors (1:100,
Sigma-Aldrich). Protein concentration was determined using a BCA
protein assay (Bio-Rad Laboratories, Hercules, CA) and 30 µg protein
was added to a 2x Laemmli buffer containing β-mercaptoethanol (5%).
Gel electrophoresis was performed, and proteins were transferred to a
PVDF membrane.
The following antibodies were obtained from Santa Cruz Biotech­
nology (Santa Cruz, CA) and used to determine protein content:
Collagen type III (1:1000), mast cell tryptase (1:20,000), CD2 (T-lym­
phocytes; 1:3000), and CD-68 (monocytes/macrophages; 1:1000). Im­
munoblots were imaged with an AlphaImager® gel documentation
system (ProteinSimple, San Jose, CA), protein bands quantified by
densitometry through use of publically available ImageJ software and
expressed relative to GAPDH (Santa Cruz Biotechnology, 1:20,000).
45
Life Sciences in Space Research 31 (2021) 43–50
2.10. Statistical analyses
Ultrasonography measured 18 heart function parameters from M-
mode and Pulsed-wave Doppler recordings of the heart and 4 parameters
from pulsed-wave Doppler of the abdominal aorta, as listed in Supple­
mentary Materials, Table S1. The measures of each these parameters
at a given time point were fitted with an analysis of covariance
comprised of radiation (0 or 0.25 Gy), GT3 (GT3 or vehicle), and their
interaction as factors. Bodyweight at euthanasia and heart rate during
ultrasonography recording were included as covariates. Others have
previously seen that measures of ultrasonography parameters may
depend on heart rate (Wu et al., 2010) and bodyweight (Pelosi et al.,
2013). For each parameter, we evaluated whether the assumptions of
normal errors and equal variances among independent groups were
reasonable. Normal assumptions were reasonable for all parameters.
However, Levene’s test for homogeneous variance indicated that for
several parameters, variances were not equal among the groups. We thus
allowed the variances among independent groups to differ for those
parameters having heterogeneous variances. Error degrees of freedom
were estimated with Kenward-Roger’s method (Kenward and Roger,
2009). Comparisons of groups were conducted with t-tests within the
analysis of covariance framework; the group means were computed at
the mean values of heart rate and bodyweight. Analyses of all other
parameters were the same as for the analysis of covariance described
above, except the covariates of heart rate and bodyweight were not
included. All tests were conducted at a 0.05 significance level.
Regarding multiple comparisons, rather than adjusting the significance
level, the positive False Discovery Rate (pFDR) was computed for all
significant results found (Storey, 2002). The ANCOVAs and ANOVAs
were conducted with the MIXED procedure in SAS/STAT software,
version 9.4 (SAS System for Windows, SAS Institute, Inc.). The pFDR was
computed in R version 3.6.2 with custom code. Statistical code and data
for analyses reported herein is available on request.
When reporting summary statistics (i.e., means ± standard deviation
(SD)), the reported means and SDs were estimated from the statistical
models since these are the values on which inferences are based. Pa­
rameters for which the assumption of homogeneous variance across
groups was reasonable, the pooled SD is reported; otherwise the SDs
were estimated (within the model) for each group.
Sample size considerations. In general, the planned sample sizes for
each of the four groups defined by the combinations of radiation dose
and GT3 administration at a specific post-irradiation time point were n
= 6, 10, and 15, depending on the parameter under investigation. With
these sample sizes, main effects of size 1.20 SD (n = 6), 0.92 SD (n = 10),
and 0.74 SD (n = 15) were detectable with 0.80 power on a 0.05 sig­
nificance level test. With the same assumptions, simple effects of size
1.71 SD (n = 6), 1.29 SD (n = 10), and 1.05 SD (n = 15) were detectable,
and interaction effects of size 2.41 SD (n = 6), 1.84 SD (n = 10), and 1.48
SD (n = 15) were detectable.
3. Results
3.1. Animal characteristics
Plasma GT3 concentrations were measured in mice sacrificed at 2
weeks after irradiation, when GT3 administrations were still ongoing.
Plasma GT3 concentrations were 0.47 ± 0.15 µM (n = 5 mice) in the
sham-irradiated animals and 0.69 ± 0.16 µM (n = 5 mice) in the irra­
diated animals. These plasma levels were not significantly different.
Across all time points examined, there was no evidence of differences
in bodyweight among the four groups (Supplemental Materials,
Table S2).
In blood chemistry values measured 9 months after total body 16O
exposure increased Cl and decreased ICA, Na, TCO2, Glucose, and BUN
compared to sham animals. GT3 mitigated these 16O induced changes in
Cl, ICA, Na, and TCO2. For BUN, levels in GT3-treated mice exposed to
A.S. Nemec-Bakk et al.
16
O radiation were also decreased from their sham irradiated peers. GT3
also demonstrated a significant increase in HB and HCT in irradiated
mice (Supplemental Materials, Table S3).
3.2. In vivo cardiac function
Ultrasonography provided 18 measures related to cardiac size and
function (described in Supplementary Materials, Table S4). At the 3
months’ time point (9 weeks after the completion of GT3 treatment),
GT3 treated animals exposed to 16O showed an increase in ejection
fraction and fractional shortening, but these increases seemed to
normalize over time (Fig. 1). Exposure to 16O caused small but signifi­
cant increases in ejection fraction and fractional shortening as measured
9 months in vehicle-treated animals. These increases may be an indi­
cation that the irradiated heart needs to compensate to maintain cardiac
output or may be an indication of left ventricular tissue remodeling.
Treatment with GT3 normalized the effects of radiation on ejection
fraction and fractional shortening in irradiated animals. No major
changes were seen in any of the other cardiac ultrasonography param­
eters (Supplementary Materials, Table S4).
Exposure to 16O and administration of GT3 did not change blood
flow parameters in the abdominal aorta (Supplementary Materials,
Table S5), suggesting that larger vessel function was not altered.
3.3. Cardiac remodeling
No differences were found in heart weight relative to tibia length to
correct for body size at any of the post-radiation time points (Supple­
mentary Materials, Table S2). If radiation fibrosis develops in the
heart, it is expected to appear late after radiation exposure. Therefore,
assessment of collagen levels in the heart was performed at 9 months
after irradiation. There was no significant difference in percentage area
of cardiac tissue occupied by collagens 9 months after radiation expo­
sure (Fig. 2).
Immunoblot analysis of collagen type III revealed a band of
approximately 75 kDa, as we previously observed in the mouse heart
(Seawright et al., 2019; S.S. Ramadan et al., 2016) that appears to be a
truncated peptide of collagen type III. As seen in our prior study (Sea­
wright et al., 2019), even though histological staining did not show an
increase in total collagen deposition in the heart, the left ventricular
content of the 75 kDa collagen type III peptide in vehicle-treated animals
was significantly increased in the 16O exposed mice over sham at 2
weeks and 3 months. The change in left ventricular content of this
peptide may be an indication of ongoing remodeling of collagen type III.
A significant interaction between radiation and GT3 at 2 weeks and 3
months indicates that GT3 mitigated the effects of radiation on the 75
kDa collagen type III peptide content (Fig. 2).
Fig. 1. Ejection fraction and fractional shortening after 16O and GT3. Cardiac ejection fraction (A) and fractional shortening (B) were determined by ultra­
sonography at 3, 5, 7, and 9 months after 16O radiation exposure. n = 11–14/group. a indicates p < 0.05 compared to time matched 0 Gy, b indicates p < 0.05
compared to time matched 0 Gy+GT3, and c indicates p < 0.05 compared to time matched 0.25 Gy. Error bars are model-estimated SDs. Horizontal lines indicate
there was a significant interaction between GT3 and radiation.
46
Life Sciences in Space Research 31 (2021) 43–50
3.4. Cardiac immune cell infiltration
In previous studies with high-dose local X-ray exposure of the heart
in rodent models, we found a correlation between cardiac mast cell
numbers and collagen deposition (Boerma et al., 2004). Therefore, we
assessed cardiac mast cell numbers as potential indicators of
radiation-induced tissue remodeling at 9 months after irradiation. There
was a significant decrease in mast cell numbers in the radiation group
compared to sham (Fig. 3). On the other hand, left ventricular mast cell
tryptase content was significantly elevated in irradiated vehicle-treated
animals over sham at 3 and 9 months. A significant interaction was seen
between GT3 and radiation at all 3 time points, indicating that GT3
mitigated the effects of radiation on left ventricular mast cell tryptase
levels (Fig. 3).
We examined CD45 positive cells as a general marker of leukocytes
(Supplementary Materials, Figure S1). In our prior study of male
C57BL/6J mice exposed to 0.25 Gy 16O (600 MeV/n), we found
consistent increases in markers of immune cells at 3 months after irra­
diation (Seawright et al., 2019), therefore, in the current study we
immunostained tissue sections for CD45, as a general marker of leuko­
cytes, and counted the number of CD45 positive cells per mm2 tissue at
the 3 months’ time point. While t-tests within the analysis of covariance
showed no significant differences between the four experimental groups,
there was a significant interaction between radiation and GT3 on the
number of CD45 positive cells that pointed to a potential mitigative
effect of GT3 (Fig. 4A).
Even though the total number of leukocytes did not differ signifi­
cantly between groups, we examined individual immune cell types by
examining left ventricular CD2 and CD68 protein levels (Fig. 4B).
Consistent with our prior study (Seawright et al., 2019), CD2 and CD68
were significantly increased in the 16O group compared to sham at 2
weeks and 3 months after irradiation. GT3 alone also significantly
increased CD2 and CD68 at 2 weeks (when GT3 administration was
ongoing) compared to sham-irradiated animals. On the other hand, GT3
treatment significantly reduced CD2 and CD68 content compared to
radiation alone at 3 months and reduced CD68 at 9 months (Fig. 4C–D).
3.5. Cardiac subcellular structure
Electron microscopy of hearts at 9 months after irradiation showed
damaged mitochondria with disrupted cristae. This damage was not
observed in mice treated with radiation and GT3 (Fig. 5).
3.6. False discovery rate
For each time point at which an outcome was measured, we made
three comparisons: (i) 0 Gy vs 0.25 Gy, (ii) 0 Gy vs 0 Gy+GT3, (iii) (0 Gy
A.S. Nemec-Bakk et al.
– 0.25 Gy) vs (0 Gy+GT3 - 0.25 Gy+GT3). Altogether, we made 336
tests; 52 were significant at the 0.05 level. The positive False Discovery
Rate was 0.285, meaning that about 15 of these 52 significant results
may be false discoveries.
4. Discussion
This study aimed to provide the first evidence to indicate whether
GT3 may be considered as a safe countermeasure against the degener­
ative tissue effects of space radiation in the heart. GT3 has long been
known as a potent protector against the acute radiation syndrome when
administered before exposure to low-LET radiation (Berbee et al., 2009;
Singh et al., 2011; Ghosh et al., 2009). However, GT3 also has several
properties that are beneficial to the cardiovascular system. GT3 is a
potent inhibitor of HMG-CoA reductase, the rate-limiting enzyme in
cholesterol biosynthesis. Interestingly, in contrast to statins, which are
direct inhibitors of HMG-CoA reductase, GT3 reduces the levels of the
enzyme by stimulating its intracellular degradation (Parker et al., 1993;
Song and Bose-Boyd, 2006). Intermediates of the cholesterol biosyn­
thesis pathway are used for post-translational modification of Rho pro­
teins that are involved in endothelial cell functions such as stress fiber
formation, expression of nitric oxide synthase, and production of cyto­
kines and growth factors (Jaffe and Hall, 2005). Therefore, inhibition of
cholesterol biosynthesis has indirect benefits to endothelial function.
47
Life Sciences in Space Research 31 (2021) 43–50
Fig. 2. Cardiac collagen levels after 16O and
GT3. Histological analysis of cardiac collagen
deposition at 9 months after irradiation (A),
representative immunoblot of left ventricular
contents of collagen type III and GAPDH at 3
months (B), and collagen type III normalized to
GAPDH at 2 weeks, 3 months, and 9 months after
radiation (C). Error bars are model-estimated
SDs. Total n = 131 animals; time points 2
weeks and 3 months had n = 10/group; and
month 9 had n = 11–14/group. a indicates p <
0.05 compared to time matched 0 Gy, b indicates
p < 0.05 compared to time matched 0 Gy+GT3,
and c indicates p < 0.05 compared to time
matched 0.25 Gy. Horizontal lines indicate there
was a significant interaction between GT3 and
radiation.
Fig. 3. Cardiac mast cells after 16O
and GT3. Cardiac mast cell numbers per
tissue area at 9 months after radiation
(A), representative immunoblot of mast
cell tryptase (MCT) and GAPDH at 3
months after radiation (B), and MCT
density normalized to GAPDH at 2
weeks, 3 months, and 9 months (C).
Error bars are model-estimated SDs.
Total n = 71 animals, with n = 5–6/
group at each time point. a indicates p <
0.05 compared to time matched 0 Gy, b
indicates p < 0.05 compared to time
matched 0 Gy+GT3, and c indicates p <
0.05 compared to time matched 0.25
Gy. Horizontal lines indicate there was a
significant interaction between GT3 and
radiation.
While some studies have shown a relation between vitamin E intake and
increased cancer rate (Klein et al., 2011), the majority of studies in
humans and animal subjects show that several forms of vitamin E,
including GT3 actually have cancer preventative properties (Jiang,
2017; Yang et al., 2020). On the basis of these combined properties of
GT3 and its apparent safety, we decided to test its effects on cardio­
vascular function and structure when administered after exposure to 16O
as a model of space radiation.
Ultrasonography demonstrated small but significant increases in
ejection fraction and fractional shortening in vehicle-treated animals at
9 months after 16O exposure. Yan et al. have also reported a small in­
crease in ejection fraction in male C57BL/6NT mice from 1 to 10 months
after a single dose of 0.15 Gy 56Fe (1 GeV/n) (X. Yan et al., 2014). We
hypothesize that this may be an indication that the irradiated heart
needs to compensate to maintain cardiac output or a consequence of
cardiac remodeling that is not yet fully understood. Treatment with GT3
mitigated the effects of radiation on ejection fraction and fractional
shortening at 9 months.
The normal heart contains low numbers of mast cells (Hellstrom and
Holmgren, 1950; Constantinides and Rutherdale, 1957). Mast cells
produce a large variety of pro- and anti-fibrogenic mediators that
regulate tissue remodeling. In our previous studies of localized cardiac
x-ray exposure, radiation-induced pathological changes such as degen­
eration and fibrosis are closely correlated with increased mast cell
A.S. Nemec-Bakk et al. Life Sciences in Space Research 31 (2021) 43–50

Fig. 4. Left ventricular protein levels of immune cell markers after 16O and GT3. Immunohistochemistry results for CD45 positive cells at 3 months after
radiation exposure (A), representative immunoblots for left ventricular CD2 and CD68 at 3 months after radiation (B), Immunoblot analysis of CD2 (C) and CD68 (D)
normalized to GAPDH at 2 weeks, 3 months, and 9 months after radiation. Error bars are model-estimated SDs. Total n = 71 animals, with n = 5–6/group at each time
point. a indicates p < 0.05 compared to time matched 0 Gy, b indicates p < 0.05 compared to time matched 0 Gy+GT3, and c indicates p < 0.05 compared to time
matched 0.25 Gy. Horizontal lines indicate there was a significant interaction between GT3 and radiation.

Fig. 5. Electron microscopy analysis of cardiac tissue after 16O and GT3.
Tissue specimens were examined at 9 months after radiation. Mitochondria in
irradiated vehicle treated mice showed disrupted cristae. Magnification: 9600
×; scale bar = 1000 nm.
numbers (Boerma et al., 2004). In the current study, mast cell numbers
as determined from histological staining were significantly decreased
after radiation exposure in vehicle-treated animals. It is possible that
whole body irradiation has an effect on overall mast cell numbers or
their ability to migrate into peripheral tissues. Nonetheless, an increase
in left ventricular protein content of mast cell tryptase was seen in
irradiated animals at the 3 and 9 month time points. These results may
suggest that existing mast cells in the heart show increased activity in
response to 16O irradiation as part of a subclinical event, rather than an
actual accumulation of mast cells in the heart. In addition to normalizing
the radiation-induced increases in ejection fraction and fractional
shortening, GT3 administration mitigated the radiation-induced in­
creases in left ventricular mast cell tryptase, suggesting its potential to
alter adverse tissue remodeling.
To further assess tissue remodeling, we examined collagens in the
heart. In vehicle-treated animals exposed to 0.25 Gy 16O, at 2 weeks and
3 months we found an increase in left ventricular content of a 75 kDa
cleavage product of collagen type III, one of the most abundant collagens
in the heart. On the other hand, there was no change in total collagen
deposition as measured with histology. These results are in accordance
with our prior findings of collagen type III at 2 weeks and 3 months and
total collagens at 9 months in male C57BL/6J mice exposed to 0.25 Gy of
16
O (600 MeV/n) (Seawright et al., 2019). Together, these results may
indicate that while radiation fibrosis does not occur, there is active
remodeling of extracellular matrix in the heart after exposure to 16O
(600 MeV/n). In contrast, in a prior study, Yan et al. reported an increase
in cardiac collagen deposition at 10 months after exposure to protons (1
GeV, 0.5 Gy) or iron ions (1 GeV/n, 0.15 Gy) in adult male C57BL/6N

48
A.S. Nemec-Bakk et al.
mice (X. Yan et al., 2014). Differences may be due to the use of a
different mouse strain or different ions. Nonetheless, in the current
study, the effects of 16O on left ventricular contents of 75 kDa collagen
type III in GT3-treated animals was significantly lower than those in
vehicle-treated animals at 2 weeks and 3 months, again suggesting the
potential of GT3 to mitigate cardiac tissue remodeling from space
radiation.
In addition to mast cell tryptase, we examined left ventricular pro­
tein content of CD2, and CD68, markers of T-lymphocytes and mono­
cytes/macrophages, respectively. CD45 (leukocytes) cell numbers were
also assessed but there was no change seen in this marker. CD2 and
CD68 immune cell markers were increased at 2 weeks and 3 months
after radiation in vehicle-treated animals, but were normalized by GT3
treatment. Previous studies have shown an increase in the number of
CD68 positive cells at 2 and 4 weeks after 56Fe (0.15 Gy, 1 GeV/n) in
mouse models (X. Yan et al., 2014; Coleman et al., 2015). Moreover,
Tungjai et al. (Tungjai et al., 2013) showed an increase in inflammatory
cytokines in the mouse heart in response to 28Si (300 MeV/n). These
results suggest HZE exposure may only affect specific immune cells and
not the immune system as a whole. Within irradiated animals, GT3
lowered CD2 levels at 2 weeks and 3 months and CD68 levels at 3
months and 9 months. Studies have previously shown tocopherols and
tocotrienols can directly regulate signaling pathways in macrophages
(Shen et al., 2018) and gene expression in T-lymphocytes (Zingg et al.,
2013). However, the effects seen in the current study may also be due to
an indirect regulation of immune cell function by GT3.
Since GT3 is known to protect mitochondria from radiation expo­
sure, cardiac mitochondria were imaged using electron microscopy.
Previous studies have demonstrated beneficial effects of tocotrienols on
the mitochondria where a single dose of tocotrienols prevented mPTP
opening alterations in mitochondrial respiration, and mitochondrial
membrane potential (Nowak et al., 2012; Sridharan et al., 2015). GT3
has also been shown to inhibit apoptosis by preventing cytochrome c
release from the mitochondria resulting in suppressed caspase -3 and -9
activation (Makpol et al., 2012). In the current study, 16O irradiation
disrupted mitochondrial cristae but this damage was not seen in the
groups treated with GT3. This result suggests GT3 can also prevent
cardiac mitochondrial damage when exposed to HZE radiation which
has not been shown before.
Blood chemistry values were obtained at 9 months after 16O exposure
to put in context with cardiovascular function and structure. A small
increase in blood concentrations of Cl and decreases in ICA, Na and
TCO2 may be an indication of reduced kidney function in 16O exposed
mice. However, kidney dysfunction was not severe enough to elevate
BUN levels. In a prior study, exposure of male Long Evans rats to 0.5 Gy
16
O (600 MeV/n) did not alter any of these blood chemistry values at 12
months after irradiation (Sridharan et al., 2020), suggesting a species or
strain dependent effect. Tocotrienol administration cause long-term
improvements in kidney function in patients with diabetes (Koay
et al., 2021). In the current study, GT3 mitigated the effects of 16O on
blood concentrations of Cl, ICA, Na and TCO2, suggesting that GT3 may
indirectly affect the cardiovascular system by preserving kidney func­
tion. In addition, GT3 caused a small increase in HB and HCT only in
irradiated animals. In prior animal studies, a single administration of
tocotrienols caused an early decrease in HB and HCT that stabilized at
day 7 (Satyamitra et al., 2012) or had no effects on HB or HCT levels
(Qureshi et al., 2011). The biological mechanisms behind the apparent
interaction between GT3 and radiation on HB and HCT in the current
study are unknown.
The high charge and ionization density of 16O are representative of
the relative biological effectiveness of the radiation environment inside
a space craft, therefore we selected 16O as the model charged particle in
this and our prior study (Seawright et al., 2019). Although the radiation
dose of 0.25 Gy in this study is within the range of total doses experi­
enced in deep space missions, practical considerations have limited this
study to administering this dose within minutes, while the dose rates of
49
Life Sciences in Space Research 31 (2021) 43–50
HZE particles during space travel are many fold lower (Cucinotta et al.,
2003). Therefore, if GT3 is further developed as radiation countermea­
sure for space travel, it should be tested in animal models of chronic or
protracted exposures to HZE radiation. Also, while the current study
focused on male C57BL/6J mice, a widely used animal model for radi­
ation studies in the cardiovascular system (S.S. Ramadan et al., 2016;
Patties et al., 2014; Ghosh et al., 2016), there may be sex differences in
the risk for cardiovascular disease, and therefore future studies will be
required in female animals.
In conclusion, results of this study provide some evidence of mild
changes in cardiac function and remodeling after whole body exposure
to a low dose of 16O (600 MeV/n) in male C57BL/6J mice, and some
indication that administration of GT3 for 3 weeks after radiation miti­
gates these effects. If GT3 is considered as one of the radiation coun­
termeasures to be developed further for space travel, its effects should be
tested in female animals and in animals chronically exposed to simu­
lated GCR.
Declaration of Competing Interest
All authors declare no conflict of interest.
Acknowledgements
This work supported by the National Space Biomedical Research
Institute under grant RE03701 through NCC 9–58, NASA grants
80NSSC17K0425 and 80NSSC19K0437, and by the National Institute of
General Medical Sciences under grant P20 GM109005. The authors wish
to thank the BNL support group, the NSRL physicists, the UAMS
Experimental Pathology Core, and the animal care staff at UAMS and
BNL for their excellent technical support.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.lssr.2021.07.006.
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