Nutrition and Cancer

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Effects of Watermelon Powder and l-arginine

Supplementation on Azoxymethane-Induced
Colon Carcinogenesis in Rats

Keith Glenn, DawnKylee S Klarich, Milica Kalaba, Arturo Figueroa, Shirin

Hooshmand, Mark Kern & Mee Young Hong

To cite this article: Keith Glenn, DawnKylee S Klarich, Milica Kalaba, Arturo Figueroa, Shirin
Hooshmand, Mark Kern & Mee Young Hong (2018): Effects of Watermelon Powder and l-arginine
Supplementation on Azoxymethane-Induced Colon Carcinogenesis in Rats, Nutrition and Cancer,
DOI: 10.1080/01635581.2018.1490782

To link to this article: https://doi.org/10.1080/01635581.2018.1490782

Published online: 12 Sep 2018.

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NUTRITION AND CANCER
https://doi.org/10.1080/01635581.2018.1490782

Effects of Watermelon Powder and L-arginine Supplementation on

Azoxymethane-Induced Colon Carcinogenesis in Rats
Keith Glenna, DawnKylee S Klaricha, Milica Kalabaa, Arturo Figueroab, Shirin Hooshmanda, Mark Kerna, and
Mee Young Honga
a
School of Exercise and Nutritional Sciences, San Diego State University, San Diego, California, USA; bDepartment of Kinesiology and
Sport Management, Texas Tech University, Lubbock, Texas, USA

ABSTRACT ARTICLE HISTORY

Diets high in fruits and vegetables may help prevent colorectal cancer (CRC). Watermelon Received 7 October 2017
consumption may reduce CRC risk due to its concentration of L-citrulline and its role in Accepted 4 June 2018
endothelial nitric oxide (NO) production. Research suggests that increased NO levels have
tumoricidal effects. The purpose of this study was to determine the effects of watermelon
powder supplementation on aberrant crypt foci (ACF) formation, precancerous lesions, and
expression of genes associated with colon carcinogenesis. Thirty-two male Sprague-Dawley
rats were assigned into three groups: control, 0.36% L-arginine, or 0.5% watermelon powder
and injected with azoxymethane (15 mg/kg body weight). Both L-arginine and watermelon
powder groups exhibited lower total numbers of ACF and high multiplicity ACF (P < 0.01).
The watermelon powder group exhibited higher NO levels and lower 8-hydroxyguanosine
DNA damage (P < 0.05). Watermelon powder and L-arginine downregulated 8-oxoguanine
DNA glycosylase gene expression and upregulated O6-methylguanine DNA methyltransfer-
ase gene expression (P < 0.05). Cyclooxgenase-2 gene expression was lower for rats fed with
watermelon powder (P < 0.05). These results suggest that watermelon powder or L-arginine
supplementation may reduce the risk of colon cancer by suppressing ACF formation
through lowering oxidative DNA damage and inflammation, modulating DNA repair enzyme
expression, and/or enhancing NO production.

Introduction
In the United States, colorectal cancer (CRC) is the
second most common cancer in both men and
women, with 95,520 new cases of colon cancer and
39,910 cases of rectal cancer expected to be diagnosed
in 2017 (1). In 2010, CRC care in the United States
cost $14 billion with treatment projected to jump to
over $17 billion by 2020 (2). Given the substantial
morbidity, mortality, and costs associated with CRC,
methods for prevention, in part through a diet high in
fruits and vegetables are under investigation (3,4). In
light of this fact, watermelon (Citrullus lanatus) con-
sumption may be a practical nutraceutical method for
reducing CRC risk factors. Due to its high concentra-
tion of L-citrulline, a precursor to L-arginine, water-
melon supplementation can contribute to endothelial
nitric oxide (NO) production (5). Evidence suggests
that increased levels of NO have tumoricidal effects
(6). L-Citrulline is naturally abundant in watermelon,
with high concentrations of L-citrulline found in both
the flesh and the rind (7). Nitric oxide synthase
(NOS) catalyzes NO from L-arginine. Delivery of
inducible NOS (iNOS) expressing cells in xenograft
human colon cancer models increases apoptosis of
xenograft tumors (8). NO compounds have been
combined with chemotherapy regimens to successfully
combat certain cancers (9,10).
L-Citrulline functions as an antioxidant in addition
to contributing to NO production (7). Evidence sug-
gests that watermelon fruit, L-citrulline, and L-arginine
may also have anti-inflammatory roles through their
abilities to inhibit the expression of pro-inflammatory
enzyme cyclooxgenase-2 (Cox-2). Cox-2 is believed to
be involved in the pathogenesis of many diseases,
including CRC (11). Reactive oxygen species (ROS) is
generated by cellular respiration as oxygen is reduced.
ROS can be both advantageous and harmful; an
unbalanced ROS system may promote oxidative stress
and can lead to a series of pathological conditions
such as cancer (12–14). Superoxide dismutase (Sod) is

CONTACT Mee Young Hong mhong2@sdsu.edu School of Exercise and Nutritional Sciences, San Diego State University, 5500 Campanile Drive,
San Diego, CA 92182-7251.
ß 2018 Taylor & Francis Group, LLC
2 K. GLENN ET AL.

a defense enzyme produced by the body to counteract Table 1. Ingredient composition of diets fed to rats.
the deleterious effects of oxidative damage (15), and Ingredient (g/kg) Control L-Arginine Watermelon
as such, watermelon juice supplementation has been Cornstarch 143.0 139.4 140.0
Sucrose 330.0 330.0 328.0
shown to prevent a reduction in Sod levels initiated Cellulose 50.0 50.0 50.0
by high dose x-ray exposure (16). Casein 200.0 200.0 200.0
Corn oil 50.0 50.0 50.0
Numerous studies exist examining the effects Milk fat 160.0 160.0 160.0
of individual components of watermelon, such as Cholesterol 10.0 10.0 10.0
Salt mix, AIN-93G 35.0 35.0 35.0
lycopene and L-citrulline, but little research has been Vitamin mix, AIN-93 10.0 10.0 10.0
conducted to evaluate the effects of watermelon DL-Methionine 3.0 3.0 3.0
supplementation on DNA damage, inflammation, and Sodium cholate 5.0 5.0 5.0
Choline bitartrate 4.0 4.0 4.0
colon cancer risk reduction. Therefore, the purpose of TBHQ 0.03 0.03 0.03
L-Arginine HCl 0.0 3.6 0.0
this study was to determine the effects of watermelon
Watermelon powder 0.0 0.0 5.0
powder supplementation on aberrant crypt foci (ACF) Total (g) 1000.03 1000.03 1000.03
formation, precancerous lesions, and expression of TBHQ: tert-Butylhydroquinone.
genes that are associated with azoxymethane (AOM)
induced colon carcinogenesis in rats. used for mucosal scraping for gene expression ana-
lysis. For ACF analysis, colon tissue was fixed in 70%
Materials and Methods ethanol solution and stained with 0.5% methylene
blue prepared in PBS. Light microscopy (50 magnifi-
Animals, Diets, and AOM Treatment
cation) was used to determine the number of ACF
Thirty-two male 21-day-old Sprague Dawley rats were and high multiplicity ACF (HMACF; foci with 4
individually housed and raised in a research facility at AC). ACF were identified by their increased size and
San Diego State University. The facility room was on darker color as previously described (17).
a 12-h light-dark cycle and temperature and humidity
were controlled at 20–24
C and 40–45%, respectively.
8-Hydroxy-20 -Deoxyguanosine DNA Adducts
Animals were allowed an acclimation period of 2–3
days prior to commencement of the study. Animal DNA damage was determined by measuring 8-
care and experimental procedures were followed in hydroxy-20 -deoxyguanosine (8-OHdG) DNA adduct
accordance with guidelines established by the San in blood using the HT 8-oxo-dG ELISA kit II
Diego State University Institutional Animal Care and (Trevigen, Gaithersburg, MD). Anti-8-OH-dG mono-
Use Committee. clonal mouse antibody and a horseradish peroxidase
After acclimation, rats were randomly allocated conjugated secondary antibody were used. TACS-
into three groups and assigned to three diets: control, Sapphire was used as a colorimetric substrate and
0.36% L-arginine, and 0.5% watermelon powder then the absorbance was read at 450 nm.
(Table 1). The watermelon powder contained 0.36% of
L-arginine þ L-citrulline and was composed of sieved,
Nitric Oxide
freeze-dried watermelon solids manufactured by Milne
Fruit Products (Prosser, WA). After 3 weeks of Blood NO production was quantified using a nitrate/
diet, rats were injected subcutaneously with AOM nitrite colorimetric assay kit (R&D Systems, Inc,
(15 mg/kg body weight), a carcinogenic agent, at the Minneapolis, MN). Enzymatic conversion of nitrate to
beginning of weeks 4 and 5. Five weeks after the nitrite was measured via Griess Reaction that converts
second AOM injection, all rats were euthanized and nitrite into a deep purple azo compound. The colori-
the colons were harvested. Food intakes were recorded metric absorbance was read at 540 nm.
over the study and body weight was recorded on a
weekly basis.
Gene Expression
Total RNA of the scraped colonic mucosa was
ACF and HMACF Analysis
extracted for gene expression analysis using TRIzol
Excised colons were resected and divided into two (Sigma, St. Louis, MO) and reverse transcription
pieces, longitudinally. Therefore, both pieces contain was performed using Oligo (dT)12-18 primer with
proximal, mid and distal parts of colon. One half was SuperScript III Reverse Transcriptase (Invitrogen,
used for ACF enumeration and the other half was Carlsbad, CA). Quantitative real time PCR (ViiA7;

Applied Biosystems, Foster City, CA) was used to ana-
lyze mRNA levels of Cox-2, Rela (nuclear factor NF-
kappa-B p65), O6-methylguanine DNA methyltrans-
ferase (Mgmt), 8-oxoguanine glycosylase (Ogg1), Sod,
and catalase (Cat). Target gene transcription levels
were normalized to r18s expression (18).
Statistical Analysis
All data were analyzed using SPSS statistical software
(IBM, Armonk, NY). One-way ANOVA was used to
determine the effects of diet on ACF, DNA damage,
NO, and gene expression. Data were presented
as means ± SE and statistical significance was set
at P < 0.05.
Results
Body Weights, Food Intake and Water Intake
The initial body weights, final body weights, and
weight gain were not statistically different among the
control, L-arginine, and watermelon groups. No differ-
ences in food intake and water intake were observed
among the groups (data not shown).
ACF and HMACF
Both L-arginine and watermelon powder diets pre-
sented a lower total number of ACF and HMACF
(P < 0.01) (Fig. 1). In comparison to the control
group, L-arginine and watermelon powder produced
74% and 82% lower numbers of ACF, respectively.
Furthermore, L-arginine and watermelon powder
Figure 1. A: Effects of L-arginine and watermelon powder on colonic aberrant crypt foci (ACF) and B: high multiplicity ACF
(HMACF). Bars represent means ± SEs. Bars with different superscripts differ significantly at P < 0.05 (Total N ¼ 32, n ¼ 10 for con-
trol, n ¼ 11 for L-arginine, n ¼ 11 for Watermelon).
NUTRITION AND CANCER 3
produced 87% and 90% lower numbers of HMACF,
respectively.
Nitric Oxide
Serum concentrations of NO are depicted in Fig. 2A.
NO was significantly higher in rats fed the water-
melon diet as compared to the control diet (P < 0.05).
No significant differences in NO concentrations
existed between the rats fed L-arginine diet and the
control diets or between the L-arginine and water-
melon powder diet groups.
8-OHdG
Serum concentrations of DNA adduct, 8-OHdG, were
significantly lower (P < 0.05) in the rats fed water-
melon powder in comparison to the rats fed the con-
trol diet (P < 0.05) (Fig. 2B). No significant differences
in concentrations of 8-OHdG were detected between
the rats fed the L-arginine diet and control diet nor the
L-arginine diet and watermelon powder diet.
Gene Expression
Watermelon powder downregulated Cox-2 expression
as compared to control group (P < 0.05) (Fig. 3A).
Cox-2 expression in the L-arginine group was not
significantly different from the control or watermelon
powder groups. There was no significant difference in
Rela among the groups (Fig. 3B).
Gene expression of repair enzyme Ogg1 was down-
regulated significantly in both the watermelon powder
and L-arginine groups compared to the control group
(P < 0.05) (Fig. 4A). Expression of the major repair
4 K. GLENN ET AL.

Figure 2. Effects of L-arginine and watermelon powder on A: nitric oxide and B: 8-hydroxyguanosine in blood. Bars represent
means ± SEs. Bars with different superscripts differ significantly at P < 0.05 (Total N ¼ 32, n ¼ 10 for control, n ¼ 11 for L-arginine,
n ¼ 11 for Watermelon).

Figure 3. Effects of L-arginine and watermelon powder on gene expression of A: Cox-2 and B: Rela. Bars represent means ± SEs.
Bars with different superscripts differ significantly at P < 0.05 (Total N ¼ 32, n ¼ 10 for control, n ¼ 11 for L-arginine, n ¼ 11 for
Watermelon). Cox-2: cyclooxygenase 2; Rela: component of NFkB.

Figure 4. Effects of L-arginine and watermelon powder on gene expression of A: Ogg1 and B: Mgmt. Bars represent means ± SEs.
Bars with different superscripts differ significantly at P < 0.05 (Total N ¼ 32, n ¼ 10 for control, n ¼ 11 for L-arginine, n ¼ 11 for
Watermelon). Ogg1: 8-oxoguanine glycosylase; Mgmt: O6-methylguanine DNA methyltransferase.

gene Mgmt was upregulated significantly (P < 0.01) in No significant differences in expression of Ogg1 or
both the watermelon powder diet and L-arginine diet Mgmt were detected between the rats fed watermelon
groups as compared to the control group (Fig. 4B). powder versus L-arginine diet.

Figure 5. Effects of L-arginine and watermelon powder on gene expression of A: Sod and B: Cat. Bars represent means ± SEs. Bars
with different superscripts differ significantly at P < 0.05 (Total N ¼ 32, n ¼ 10 for control, n ¼ 11 for L-arginine, n ¼ 11 for
Watermelon). SOD: superoxide dismutase; CAT: catalase.
Upregulated Sod expression was detected for the
L-arginine group compared to control group
(P < 0.05) (Fig. 5A). No differences were found
between the L-arginine and watermelon powder
groups or between the watermelon and control
groups. Although there was a trend for upregulation
of Cat between the watermelon powder and L-arginine
groups compared to the control group (P ¼ 0.086),
there was no significant difference in Cat expression
among the three groups (Fig. 5B).
Discussion
The onset of CRC is in part caused by environmental
factors, yet studies suggest that the inclusion of fruits
and vegetables in the diet can potentially reduce the
occurrence of cancer (19,20). In the present study, sig-
nificantly lower numbers of ACF and HMACF were
observed in animals fed either L-arginine or water-
melon powder diets as compared to a control diet
after AOM treatment. Similar results were detected in
a study by Boateng et al. (21), who reported that a
20% watermelon juice diet reduced ACF formation by
86% as compared to the control group.
A potential mechanism for ACF reduction may be
attributed to the role of L-citrulline and L-arginine in
NO production via the NOS pathway. As previously
mentioned, L-citrulline is a main component of water-
melon and a precursor to L-arginine, which plays a
key role in NO production. Increased serum levels of
L-citrulline and L-arginine have been detected after
watermelon juice consumption (22–23). A study by
Schleiffer et al. (24) indicated a 47% increase in ACF
formation when the NOS pathway was inhibited by
NG-nitro-L-arginine methyl ester. L-Arginine is a
NUTRITION AND CANCER 5
common precursor for both NO and polyamines and
the inhibition of the NOS pathway thus increases the
rate of polyamine synthesis. Tumor development is
associated with heightened levels of polyamines,
formed by a key enzyme, arginase ornithine-
decarboxylase (ODC) (25–27). Data from Schleiffer
et al. (24) suggest that ODC expression is regulated by
NO levels, and increased NO levels suppress ODC
activity and potential tumorigenesis. The present study
showed that high NO levels resulting from water-
melon powder supplementation potentially downregu-
late ODC in a similar manner as described by
Schleiffer et al. (24), thus preventing formation of
ACF and potential advancement to CRC. Future stud-
ies might focus on altered ODC activity as a function
of watermelon powder supplementation.
In the present study, NO levels were higher in the
L-arginine and watermelon powder groups compared
to the control group, which may have contributed to
the lower numbers of ACF. These findings are con-
sistent with a study by Yeh et al. (28), which theorized
that tumor progression was reduced due to endogen-
ous NO production by tumor and host cells as a
result of dietary arginine supplementation. A study by
Yerushalmi et al. (29) contradicts these findings,
demonstrating that L-arginine derived inducible NO
promotes tumorigenesis in a mice model. NO plays a
dual role in stimulating tumor growth under certain
conditions while inhibiting neoplasia and angiogenesis
under other conditions (30). More research is needed
to further define this dual role.
Inflammation is a key risk factor in the occurrence
of CRC as chronic inflammation promotes the induce-
ment of gene mutations, inhibition of apoptosis, and
the stimulation of cell proliferation and angiogenesis
6 K. GLENN ET AL.
(31,32). Inflammation can also trigger non-genetic
influences on gene expression that are associated with
carcinogenesis. Cox-2 and Rela are key factors in the
inflammatory process that provide a link between
inflammation and CRC; both have been widely
researched for their potential roles in chemopreven-
tion (33). The inflammatory process includes immune
cells entering colonic epithelial cells and activating
Rela, which in turn stimulates pro-inflammatory gene
expression of Cox-2 (31,34). Continuous activation of
inflammatory Rela promotes colonic tumor develop-
ment. Cox-2 gene expression results in oxidative stress
early in the inflammation process and is associated
with the progression of CRC (35,36). Due to this asso-
ciation, Cox-2 inhibitors have been found to have che-
mopreventive effects if administered early in CRC
(37). NO has also been indicated as a modulator for
Cox-2 expression. The present findings indicate that
watermelon and L-arginine may have contributed to
increased NO and decreased COX-2 expression within
the colon mucosa. Possible links between the concen-
tration of NO and COX-2 expression have been
explored in previous research, demonstrating both
inhibitory and stimulatory effects of NO on the
expression of COX-2 (38). In a study by Shin et al.
(39), examining COX-2 expression in systems treated
with soy-isoflavones, an increase in both NO and
COX-2 was observed as well an increase in prosta-
glandin E2. In contrast, a study by Clancy et al. dem-
onstrated an inactivation of COX-2 isoform by
increased levels of NO. Furthermore, the study by
Clancy et al. (40) suggests that NO wields divergent
effects on the constitutive and inducible COX iso-
forms by activating COX-1 and inactivating COX-2.
Further studies are warranted to determine the mech-
anisms by which NO effects COX enzyme expression.
Oxidative stress initiated by AOM increases
oxidative DNA damage and colon carcinogenesis in
animals. Oxidative DNA damage is marked by the
expression of 8-OHdG (41–43). In the present study,
results of the AOM treatment were consistent with
previous findings as levels of 8-OHdG were elevated.
Compared to the control, the watermelon group
exhibited significantly lower levels of 8-OHdG, indi-
cating decreased levels of oxidative DNA damage, pos-
sibly due to an enhanced DNA repair system resulting
from watermelon supplementation. Mgmt has a cen-
tral role in DNA stability as it repairs the mutagenic
DNA lesion O6-methylguanine back to guanine,
preventing errors during replication and transcription
of DNA (44). Tumor cells from rat colon mucosa can
decrease levels of Mgmt (45). This is consistent with
the present study as the control group had signifi-
cantly lower expression of Mgmt as compared to both
the L-arginine and watermelon powder groups. Ogg1
is a major repair enzyme that excises the 8-oxo-
20 deoxyguanosine adduct via the base excision repair
pathway (46). Ogg1 levels of the control group were
significantly higher relative to both the L-arginine and
watermelon powder groups, demonstrating that a
lower level of oxidative DNA damage may be associ-
ated with lower induction of Ogg1 levels. The increase
in Mgmt and decrease of Ogg1 in both treatment
groups may be associated with lower DNA damage in
the colon mucosa resulting in lower ACF formation.
AOM injections occurred at weeks 4 and 5, thus,
ingestion of watermelon powder and L-arginine
occurred before, during, and after these carcinogenic
administrations. As a result, the presumed beneficial
effects on colon carcinogenesis could be due to an
anti-initiating effect, an anti-promoting effect, or a
combination of the two. Further investigation is
warranted to specifically determine the onset of these
putative benefits. In addition, our future studies will
attempt to determine if the observed changes in gene
expression as a function of watermelon is concurrent
with the alteration in protein concentration.
Enzymes Sod and Cat help protect cells from oxi-
dative damage caused by ROS and potential DNA
damage leading to carcinogenesis (47,48). A signifi-
cant increase in Sod was observed in the L-arginine
group compared to the control group, with no differ-
ences in Sod between the L-arginine and watermelon
powder groups. This may be a contributing factor in
the lower oxidative damage observed in the present
study. Mohammed et al. (16) found significant
increases in Sod enzyme activity in a study examining
the effects of watermelon juice consumption in a mice
model with induced oxidative damage. A separate
study by Ren et al. (49) observed a beneficial effect of
L-arginine supplementation on Sod levels in an
induced colitis mouse model. The positive results
were associated with a number of colonic signaling
pathways, including NFjB.
Beneficial effects have also been correlated with the
phytochemicals carotenoids and phenolic acids, other
major components in watermelon. Notable carote-
noids include lycopene, phytoene, beta-carotene,
lutein and neurosporene (50). A matrix with other
bioactive constituents in watermelon may also
contribute to the anti-cancer and anti-inflammatory
effect.
In conclusion, these results suggest that watermelon
powder or L-arginine consumption may suppress ACF

formation through lowering oxidative DNA damage
and inflammation, modulating DNA repair enzyme
expression, and enhancing NO production. L-citrulline
may be at least partially responsible for these effects
of watermelon, as it is a precursor to L-arginine, an
amino acid that plays a crucial role in NO production.
More research is needed to investigate any dose
dependent effects of watermelon powder on ACF for-
mation, as well as the effects of watermelon powder
on various stages of tumor development.
Acknowledgment
The authors thank Milne Fruit Products for providing the
watermelon extract powder.
Disclosure Statement
The authors declare that they have no conflicts of interest.
There is no conflict of interest with the National
Watermelon Promotion Board.
Funding
The study was funded by The US National Watermelon
Promotion Board.
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