This study examined the effects of chronic endurance exercise on doxorubicin (DOX)-induced damage in the thymus gland and thymocytes (T-cells). Rats were divided into groups that were sedentary or underwent treadmill training for 10 weeks, followed by injections of either saline or different doses of DOX. Three days later, thymic mass, viable T-cell count, and lipid peroxidation levels were analyzed. Chronic exercise decreased lipid peroxidation following DOX treatment but did not prevent reductions in thymic mass or T-cell numbers. This suggests that exercise elevates antioxidant defenses in the thymus to reduce oxidative stress from DOX, though it does not fully protect the

1. Integrative Cancer Therapies
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Research Articles
Introduction
The anthracycline doxorubicin (DOX; trade name:
Adriamycin) is a frequently prescribed chemotherapy agent
used in the treatment of a number of cancers. Unfortunately,
negative side effects in healthy tissues commonly occur and
limit dosage. Beyond toxicities seen in cardiac and hepatic
tissues, DOX has been shown to induce thymic involution
with concurrent decreases in local thymocyte (T-cell)
number.1-3
When lymphocytes are exposed to DOX in vitro,
elevation in markers of cellular apoptosis is observed.4
In
addition to prevention of DNA synthesis, the antitumor
effects of DOX may be attributed to the generation of reac-
tive oxygen species (ROS).5
ROS manifest in the form of
superoxide, hydrogen peroxide and hydroxyls, which can
damage membranes and initiate cell death.
Acute, exhaustive exercise elicits glucocorticoid produc-
tion and decreases in circulating lymphocytes, with subse-
quent reduction in thymus gland size.6
Chronic, submaximal
exercise, however, does not provoke these adverse impacts
but stimulates favorable elevations in levels of free-radical-
scavenging enzymes.7,8
Likewise, prolonged aerobic train-
ing prior to DOX exposure has shown to increase antioxidant
expression and activity in cardiac tissue.9-14
It is possible
that similar adaptations to chronic endurance training may
also mitigate DOX effects through increased handling of
ROS in the thymus.
Cancer patients receiving chemotherapy exhibit
decreased immune responses following treatment and may
be more susceptible to disease.15
Because DOX use is
widely prescribed in cancer patients, chronic endurance
training prior to treatment may be useful in enhancing
immune systems of this population. The effect of submaxi-
mal, chronic exercise prior to DOX administration on the
thymus, however, remains unknown. Therefore, the pur-
pose of this study was to examine the effects of chronic,
submaximal endurance exercise prior to acute DOX admin-
istration on thymus size, viable T-cell number, and thymic
levels of lipid peroxidation. It was postulated that chronic
endurance exercise would attenuate thymic deficiencies
associated with DOX administration.
617014ICTXXX10.1177/1534735415617014Integrative Cancer TherapiesQuinn et al
research-article2015
1
University of Northern Colorado, Greeley, CO, USA
Corresponding Author:
David Hydock, School of Sport and Exercise Science, University of
Northern Colorado, 501 20th Street, Greeley, CO 80639, USA.
Email: David.Hydock@unco.edu
Effects of Chronic Endurance Exercise on
Doxorubicin-Induced Thymic Damage
Colin J. Quinn, PhD1
, Patrick D. Burns, PhD1
, Noah M. Gibson, MS1
,
Alex Bashore, BS1
, Reid Hayward, PhD1
, and David S. Hydock, PhD1
Abstract
The use of prior exercise training has shown promise in minimizing doxorubicin (DOX)-induced physical impairments.
The purpose of this study was to compare changes in thymus mass, thymocyte (T-cell) number, and tissue peroxidation
following chronic endurance exercise and DOX treatment in the rat. The thymus mass, number of viable T-cells, and
levels of malondialdehyde and 4-hydroxyalkenals (MDA+4-HAE) were compared 3 days post-injection between rats
assigned to the following treatment conditions: (a) 10 weeks of endurance training, followed by a saline injection 24 hours
after the last training session (TM+SAL); (b) treadmill training as above, followed by a single, bolus 10-mg/kg injection of
DOX (TM+10); (c) treadmill training with 12.5 mg/kg of DOX (TM+12.5); (d) sedentary (without exercise) and a saline
injection (SED+SAL); (e) sedentary with 10 mg/kg of DOX (SED+10); and (f) sedentary with 12.5 mg/kg (SED+12.5).
Thymic mass and T-cell numbers significantly decreased following DOX injections. TM rats exhibited significantly less lipid
peroxidation compared with paired-dose SED groups. TM+10 did not significantly differ from SED+SAL in thymic levels of
lipid peroxidation. We conclude that chronic endurance exercise decreases levels of lipid peroxidation in the thymus seen
with acute DOX treatment.
Keywords
Adriamycin, anthracycline, lipid peroxidation, physical activity, thymocyte
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2. 2 Integrative Cancer Therapies 
Materials and Methods
Animals
A total of 48 male, 10-week-old Sprague-Dawley rats
(Harlan, Indianapolis, IN) were housed in the University of
Northern Colorado Animal Research Facility in 12-hour
light/dark conditions at thermoneutrality (21.0°C ± 1.0°C)
and fed rodent lab chow (Harlan Teklad 2016) and distilled
water ad libitum. The animal use protocol was approved by
the University of Northern Colorado Animal Care and Use
Committee and is in accordance with the Animal Welfare
Act. Initially, rats were randomly assigned to treadmill
exercise (TM, n = 23) or sedentary (SED, n = 25) conditions
for 10 weeks. Following the activity period, rats were ran-
domly assigned to receive either DOX or saline injections.
Experimental Design
Rats assigned to TM trained for 10 weeks using a progres-
sive training protocol on a motorized treadmill (Table 1).
An initial acclimation to the treadmill was performed at
week 0. Exercise duration and intensity (initial setting: 25
m/min, 0% slope, 20 min/session, 5 d/wk) gradually
increased until week 8, when it reached final conditions (30
m/min, 18% slope, 60 min/session, 5 d/wk).Acontrol group
(SED+SAL/10/12.5) remained sedentary (normal cage
activity) for 10 weeks. TM received bolus DOX (Sagent:
Schaumburg, IL) or saline injections 24 hours after the last
training session. Injections were administered intraperito-
neally according to milligram per kilogram body weight.
TM+10 and SED+10 animals received 10 mg/kg of DOX.
TM+12.5 and SED+12.5 animals received 12.5 mg/kg of
DOX. TM+SAL and SED+SAL animals received equiva-
lent volumes of saline. Animals were anesthetized with
heparinized sodium pentobarbital 72 hours after injections.
After a tail-pinch reflex was absent, animals were killed
humanely by aortic exsanguination, and the thymus was
excised.
Tissue Preparation
Rat thymus glands were isolated and maintained in ice-cold
phosphate buffered solution (PBS 1×) supplemented with
glucose. After cleansing and wet weight recording, the thy-
mus was briefly exposed (<10 s) to hypotonic PBS (10×) to
lyse any remaining red blood cells on the tissue and quickly
restored to 1× PBS. Lymphocytes were expressed from the
thymus by gently teasing organs between frosted-edge glass
slides into a cell culture dish containing 5 mL PBS. Slides
were rinsed with PBS over the collecting dish to ensure a
complete thymocyte delivery. After gentle mixing, 10 µL of
thymocytes in PBS were added to a polypropylene tube
with an equal volume of trypan blue (Sigma-Aldrich, St
Louis, MO). The blend of thymocyte solution/trypan blue
was thoroughly mixed via pipette and centrifuged before
being added to a hemocytometer slide for manual viable
cell count. An upright 10× microscope and CellSens soft-
ware (Olympus, Tokyo, Japan) were used to examine the
hemocytometer slide. Exclusion of trypan blue indicated
viable status, whereas inclusion staining denoted perfora-
tion of cell membranes in nonviable thymocytes.
Immediately after T-cell collection, thymus tissue was flash
frozen in liquid nitrogen and stored at −80°C until biochem-
ical analysis.
Lipid Peroxide Determination
Lipid peroxidation was assessed in thymic tissue as an indi-
cator of DOX-induced cellular oxidative damage.
Malondialdehyde and 4-hydroxyalkenals (MDA+4-HAE)
were determined using a commercially available kit (Oxis
International, Inc, Portland, OR). Thymic tissues were
homogenized in RIPA buffer and centrifuged at 3000g for
10 minutes. Supernatant was removed, and protein concen-
tration was analyzed using the Bradford protocol.16
Total
protein concentrations were standardized, and a 200-µL ali-
quot of each sample was added to 650 µL of N-methyl-2-
phenylinodole and briefly vortexed. Next, 150 µL of
methanesulfonic acid was added, vortexed, and incubated
(45°C) for 60 minutes. Following centrifugation at 15,000g
for 10 minutes, supernatant was transferred to a cuvette, and
absorbency was measured at 586 nm. A standard curve of
provided reagents and different concentrations of MDA
provided a linear regression analysis for MDA+4-HAE at r²
= 0.99990. All samples were run in duplicate, and if absor-
bency differed by >5%, samples were reassayed.
Statistical Analysis
All data are presented as mean ± standard error of the mean.
A 2-factor (Activity × Drug) ANOVA was performed to
determine differences in physical characteristics (body and
tissue masses), viable T-cell numbers, and MDA+4HAE
concentrations. When a significant difference was detected
between groups, a Bonferroni post hoc analysis determined
which groups differed. Significance was set at a P < .05
level. Statistical analyses were performed using the Prism
software package (GraphPad, LaJolla, CA).
Results
Animal Characteristics
All TM rats completed exercise training. Animal character-
istics are presented in Table 2. Body mass (BM) from the
SED+12.5 group was significantly less than that in SAL
groups. Rats receiving DOX displayed significant reduction
in BM, except TM+12.5. This group did not significantly
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3. Quinn et al 3
differ from the TM+SAL group. All rats receiving DOX
possessed significantly lower thymus masses than SAL ani-
mals, as seen in Figure 1. Thymus mass relative to BM was
also significantly lower in rats receiving DOX.
Thymocyte Viability
Figure 2 illustrates viable T-cell numbers between groups.
There was a significant drug effect observed (P = .0107) with
DOX administration decreasingT-cell numbers. No significant
exercise effect or interaction was observed, and post hoc test-
ing did not reveal any between-group differences (P > .05).
Lipid Peroxidation
As shown in Figure 3, a significant drug effect was observed
in MDA+4HAE levels (P < .0001), indicating that DOX
treatment elevated oxidative stress levels. A significant
activity effect (P < .0001) was seen, with chronic exercise
suppressing levels of lipid peroxidation. Additionally, a sig-
nificant interaction (P = .0385) was observed. TM+10
exhibited significantly less MDA+4HAE than SED+10 and
was not significantly different from SED+SAL. TM+12.5
thymus had significantly less MDA+4HAE than the
SED+12.5 as well.
Discussion
To our knowledge, this is the first study to examine the
effects of chronic exercise prior to DOX treatment on the
thymus. We examined changes in tissue mass, local T-cell
count, and MDA+HAE. Our main finding was that chronic
endurance exercise significantly reduced levels of oxidative
stress following DOX injections. This finding is in agree-
ment with previous studies suggesting that chronic exercise
elevates antioxidant enzyme levels and activity. However,
exercise did not prevent thymic involution and thymocyte
loss. Although T-cell number was not significantly different
between TM and SED animals receiving 10 mg/kg
(P = .1701), a trend of higher viable T-cell count with endur-
ance exercise was noted.
DOX is an antineoplastic agent used in a wide variety of
cancers. In spite of its efficacy combating cancerous cells,
accompanying side effects limit dosing. The most recog-
nized side effect accompanying DOX administration is car-
diotoxicity because DOX accumulates in cardiac cells and
undergoes redox cycling resulting in the formation of
ROS.5,17
ROS-associated damage occurs as peroxidizing of
Table 2. Physical Characteristics of Subjects.a
SED+SAL
(n = 8)
SED+10 mg/kg
(n = 8)
SED+12.5mg/kg
(n = 7)
TM+SAL
(n = 9)
TM+10 mg/kg
(n = 9)
TM+12.5 mg/kg
(n = 7)
Body mass (g) 432.8 ± 6.3 401.0 ± 10.8 375.9 ± 7.6b,c
441.9 ± 9.2 394.9 ± 16.3 422.6 ± 16.1
Δ Body mass (g) 2.8 ± 4.9 −38.7 ± 3.8b,c
−32.1 ± 8.5b,c
0.8 ± 2.9 −44.0 ± 6.4b,c
−26.9 ± 7.1b
Thymus mass (g) 265.7 ± 13.6 123.0 ± 10.3b,c
122.8 ± 15.7b,c
258.8 ± 17.6 113.1 ± 13.6b,c
116.4 ± 9.9b,c
Thymus/Body (mg/kg) 614.8 ± 35.3 282.7 ± 43.4b,c
319.1 ± 136.4b,c
556.5 ± 46.4 288.8 ± 29.9b,c
275.0 ± 20.3b,c
Abbreviations: SED, sedentary; SAL, saline treated; TM, treadmill trained; 10 mg/kg, 10 mg/kg doxorubicin treated; 12.5 mg/kg, 12.5 mg/kg doxorubicin
treated; Δ Body mass, change in body mass before and after injections.
a
Data are mean ± standard error of the mean.
b
Significantly different compared with sedentary/saline group (P < .05).
c
Significantly different compared with exercise/saline group (P < .05).
Figure 1. Thymus size comparison: A. SED+DOX; B. SED+SAL.
Abbreviations: SED, sedentary; DOX, doxorubicin; SAL, saline treated.
Table 1. Progressive Treadmill Training Protocol.
Week
1 2 3 4 5 6 7 8 9 10
Speed (m/min) 25 25 25 30 30 30 30 30 30 30
Incline (%) 0 0 0 3 6 9 12 15 18 18
Duration (minutes) 20 30 30 60 60 60 60 60 60 60
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4. 4 Integrative Cancer Therapies 
the lipid constituting membranes, at the cellular, mitochon-
drial, and nuclear levels.18
Additionally, DOX intercalates
DNA and stabilizes topoisomerases, preventing synthesis
and replication. DOX-affected cells may undergo apoptotic
events with eventual cell death. Although cardiac tissue has
received a majority of the attention regarding associated
toxicity, DOX has also been shown to induce thymic invo-
lution and T-cell senescence.1
Additionally, isolated rat thy-
mocytes exposed to DOX undergo DNA fragmentation,
with subsequent increases in ROS scavenging enzyme
activities of superoxide dismutase (SOD) and catalase
activities.4
Although elevated antioxidant enzymes could
not prevent apoptosis, the stimulation of antioxidant
enzymes suggests that apoptosis may be partly a result of
free radical formations. In vitro exposure of murine lym-
phocytes to DOX induces rapid DNA degradation in mostly
noncycling cells, with greater cell death dependent on DOX
concentrations.19
The immune system functions to identify and eliminate
foreign antigens that enter the body.20
Leukocytes, or white
blood cells, originate in bone marrow, but mature in organs,
including the thymus, spleen, lymph nodes, and peripheral
lymphoid organs. Leukocytes that act directly on antigens
are B- and T-cells, with B-cells creating antibodies specific
to antigens and T-cells destroying targeted cells. B-cells
mature in bone marrow, whereas T-cells mature in the thy-
mus. T-cells, or thymocytes, are cytotoxic (CD8+
), helper
(response-enhancing, CD4+
), or regulatory (Treg) cells.
Various conditions of stress have been shown to decrease
T-cell number and lead to thymic involution, including anx-
iety, physical stress, and heat/cold exposure.21-24
Physical stress, such as exercise, may alter immunologi-
cal capacity. Nieman’s25
J-shaped model of exercise and
immunity suggests that exercise can enhance or reduce
immune function depending on frequency, duration, and
intensity of exercise performed. Evidence suggests that
strenuous acute bouts of exercise decrease circulating lym-
phocyte number while increasing apoptosis, as evidenced
by DNA fragmentation.26,27
Conversely, submaximal exer-
cise in mice demonstrates fewer apoptotic and higher num-
bers of viable T-cells compared with sedentary controls.28
Although peroxidation of lipids of cellular and mitochon-
drial membranes have been primarily implicated in the
induction of apoptosis, isolated rat nuclei exposed to DOX
also exhibit membrane peroxidation.29
In addition to ele-
vated ROS following strenuous exercise, increased gluco-
corticoids, intracellular Ca2+
, and inflammatory signals may
induce apoptosis during times of suppressed immune func-
tion.30
A rise in glucocorticoid release and intracellular Ca2+
levels may further induce apoptosis in lymphocytes.31
Glucocorticoids released in response to exercise, such as
cortisol, have been shown to induce pyknosis and DNA
fragmentation in isolated thymocytes.32
In accordance with the J-shaped model, studies examin-
ing response to chronic and acute endurance exercise have
revealed opposing lymphocytic effects. Chronic exercise
improved thymic structural quality and circulating lympho-
cyte populations, whereas acute, exhaustive bouts had
Figure 2. Graphical representation of viable T-cell number:
data are mean ± standard error of the mean. Significant drug
effect (P = .0107); no activity or interaction effect (P > .05).
Abbreviations: SED, sedentary; TM, treadmill trained; SAL, saline
treated; 10 mg/kg, 10 mg/kg doxorubicin treated; 12.5 mg/kg, 12.5 mg/kg
doxorubicin treated.
Figure 3. Graphical representation of lipid peroxidation in the
thymus: data are mean ± standard error of the mean.
Abbreviations: SED, sedentary; TM, treadmill trained; SAL, saline
treated; 10 mg/kg, 10 mg/kg doxorubicin treated; 12.5 mg/kg, 12.5 mg/kg
doxorubicin treated; DOX, doxorubicin.
a
Significantly different as compared with sedentary/saline group (P < .05).
b
Significantly different as compared with exercise/saline group (P < .05).
c
Significantly different as compared with sedentary/DOX 10-mg/kg group
(P < .001).
d
Significantly different as compared with sedentary/DOX 12.5-mg/kg
group (P < .01).
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5. Quinn et al 5
negative effects on the thymus, lymphocyte populations,
and other immune organs.33
Specifically, thymic tissues
express significantly elevated membrane lipid peroxidation
after mice were acutely run to exhaustion.27
Furthermore,
T-cells exhibited higher levels of intracellular Ca2+
and lipid
peroxidation after exercise.34
Navalta et al35
demonstrated
that exercise intensity affects circulating lymphocyte apop-
tosis, with significance occurring above 60% of VO2max in
untrained human subjects performing incremental treadmill
tests to exhaustion.
To determine training effects on lymphocyte apoptosis in
response to chronic training, Mooren et al36
compared
trained runners against poorly trained individuals and found
cell death induced by exercise in the poorly trained indi-
viduals. Enhanced serum antioxidant enzyme production
with chronic wheel running (10 months) in mice demon-
strated reduced ROS-induced apoptosis in immune cells.37
An increase in antioxidant enzymes through chronic exer-
cise training may attenuate ROS-induced damage seen in
lymphocytes.7
Additionally, 8 weeks of treadmill- and
swim-training induced significantly higher levels of CD4+
and CD8+
T-cells in the thymus of Sprague-Dawley rats.38
Although acute, exhaustive exercise may elevate glucocor-
ticoids, which initiate lymphocytic cell death, chronic sub-
maximal training may not trigger such levels.
It has been long known that exhaustive exercise in rats
induces significant thymic atrophy.21
Additionally, studies
demonstrate that lymphocyte apoptosis occurs immediately
following high-intensity exercise.26
Using a submaximal,
chronic training program, however, may enhance antioxi-
dant enzyme concentrations and alleviate ROS-related lym-
phocyte apoptosis without the negative side effects seen
with exhaustive bouts. Sugiura et al39
determined that 12
weeks of forced, submaximal running in mice (15 m/min)
significantly increased spleen and liver mass while slightly
enlarging thymic tissue compared with sedentary animals.
Using a swim-training model for 8 weeks, Pereira et al7
demonstrated that lipid peroxidation levels were signifi-
cantly decreased in thymus tissues versus sedentary con-
trols. Additionally, citrate synthase and GPX levels were
significantly elevated. Short-term exercise (3 weeks) in
hemodialysis patients does not restore T-cell numbers com-
parable to normal healthy individuals, but T-cell activity is
elevated when compared with sedentary hemodialysis
patients.40
It is suggested that chronic, moderate exercise
may increase total T-cell number with amplified activity.41
This study did not examine the effects of exercise intensity
with DOX treatment on thymic changes.
The thymus naturally reduces the size and total number
of T-cells in an age-dependent fashion, leading to decreased
immune resistance, referred to as immunosenescence.1,42
As
humans age, the number of cytotoxic CD8+
T-cells tend to
decrease with senescence. Moderate exercising older adults
exhibit greater proportions of CD8+
T-cells when compared
with sedentary individuals.43
Patients receiving chemother-
apy also have a suppressed immune system. As demon-
strated in this study, acute DOX treatment significantly
reduces size of thymic tissue and reduces viable T-cell num-
bers. There was an exercise effect, with endurance-trained
animals presenting significantly less thymic lipid peroxida-
tion when compared with sedentary paired groups.
Furthermore, the level of MDA+4HAE in endurance-
trained rats receiving 10 mg/kg DOX (TM+10) was not sig-
nificantly different from that in sedentary/saline animals
(SED+SAL). A significant drug effect was noted with DOX
treatment in total T-cell number, and prior exercise did not
significantly attenuate these losses. The addition of DOX-
induced thymus dysfunction may further compromise the
ability of patients to stave off further infections experienced
during treatment and shortly thereafter. A prior endurance
exercise program may reduce the thymic-related losses in
size and T-cell number.
Clinical implications of this study suggest that chroni-
cally trained individuals may demonstrate greater
immune response following chemotherapy treatment
than sedentary counterparts. Clearly, this study demon-
strates that DOX treatment results in significant thymic
atrophy. Production of naïve T-cells is governed by thy-
mus output and not replication of the circulating cell
pool.44
Thymic involution, or atrophy, results in decreased
T-cell development and thymopoiesis.45
Conversely,
increased size of thymus may complement T-cell produc-
tion and new antigen resistance in the time following
treatment. DeNardo et al46
suggest that, in addition to
increased macrophage number, an abundance of T-cells
indicate greater survival rate among breast cancer
patients. Additionally, Feng et al47
showed that mice in
an oxidative stress model exhibit significantly less T-cell
proliferation than controls. Exercise preconditioning
lowered markers of oxidative stress and may attenuate
decreased T-cell production following DOX treatment.
The optimal cumulative dose of DOX for induction of
congestive heart failure (CHF) has been established at
12.45 mg/kg in rats.48
The doses used (10 and 12.5
mg/kg) in this study may elicit outcomes similar to those
experienced in clinical settings. However, clinical admin-
istration of DOX is typically delivered in small doses
over the course of treatment. The single bolus injection
strategy is a limitation of the present study. Viable T-cell
numbers in this study may have been overestimated in
samples because of cells undergoing early stages of
apoptosis. During early apoptosis, cell membranes
remain intact and do not allow trypan blue entry into
cells. The early-apoptotic T-cells were not distinguished
in this study. Although this study did not examine circu-
lating thymocytes, animals had not exercised for 3 days
after injection, and lymphocyte circulation typically ele-
vates following stressful conditions.
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6. 6 Integrative Cancer Therapies 
Conclusion
Data from the current study suggest significant decreases in
thymic lipid peroxidation with chronic endurance exercise
prior to DOX administration. Additionally, exercise precon-
ditioning may have contributed to an increasing trend in
T-cell count. Individuals who aerobically exercise at sub-
maximal intensities prior to chemotherapy may offset thy-
mocytic depressions typical with DOX treatment. The
observed depression in levels of MDA+4HAE may be
attributed to elevated antioxidant enzyme levels and activ-
ity associated with chronic endurance exercise training.
Acknowledgments
The authors wish to thank Gregory DeKrey for his technical con-
tribution to this investigation.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
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