Drinking Water with Red Beetroot Food Color Antagonizes Esophageal
Carcinogenesis in N-Nitrosomethylbenzylamine-Treated Rats
John F. Lechner,1,2,*Li-Shu Wang,1,*Claudio M. Rocha,1Bethany Larue,1Cassandra Henry,1
Colleen M. McIntyre,1Kenneth M. Riedl,3Steven J. Schwartz,3and Gary D. Stoner1
1Comprehensive Cancer Center and3Department of Food Science, College of Agriculture,
The Ohio State University, Columbus, Ohio; and2Maine Center for Toxicology and Environmental Health,
University of Southern Maine, Portland, Maine, USA
inhibition of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. Rats were treated with NMBA and
given either regular water ad libitum or water containing 78mg=mL commercial red beetroot dye, E162. The number of
NMBA-induced esophageal papillomas was reduced by 45% (P<.001) in animals that received the food color compared to
controls. The treatment also resulted in reduced rates of cell proliferation in both precancerous esophageal lesions and in
papillomas of NMBA-treated rats, as measured by immunohistochemical staining of Ki-67 in esophageal tissue specimens.
The effects of beetroot food color on angiogenesis (microvessel density by CD34 immunostaining), inflammation (by CD45
immunostaining), and apoptosis (by terminal deoxynucleotidyl transferase dUTP nick end-labeling staining) in esophageal
tissue specimens were also determined. Compared to rats treated with NMBA only, the levels of angiogenesis and inflam-
mation in the beetroot color-consuming animals were reduced, and the apoptotic rate was increased. Thus, the mechanism(s)
of chemoprevention by the active constituents of red beetroot color include reducing cell proliferation, angiogenesis, and
inflammation and stimulating apoptosis. Importantly, consumption of the dye in the drinking water for a period of 35 weeks
did not appear to induce any overt toxicity. Based on the fact that red beetroot color contains betanins, which have strong
antioxidant activity, it is postulated that these effects are mediated through inhibition of oxygen radical-induced signal
transduction. However, the sum of constituents of E162 has not been determined, and other components with other mech-
anisms may also be involved in antagonizing cancer development.
This study was undertaken to determine if the oral consumption of red beetroot food color would result in an
KEY WORDS:?red beetroot dye?esophageal carcinogenesis?rat
cell papillomas in the rat esophagus.1,2We have shown
that freeze-dried black raspberries (Rubus occidentalis),
which contain several anticancer agents such as ellagic
acid, b-carotene, a-carotene, lutein, gallic acid, ferulic
acid, p-coumaric acid, quercetin, b-sitosterol, stigmasterol,
kaempferol, and anthocyanins, will reduce the number of
NMBA-induced esophageal papillomas in rats when added
to the diet at concentrations of 5% and 10%.3–6When added
at 20% of the diet, however, freeze-dried black raspberries
do not further reduce papilloma multiplicity in this model.5,6
benzylamine (NMBA) is a potent inducer of squamous
We have questioned, therefore, whether the esophageal
tumorresponseto NMBAcould befurther reducediftherats
consumed other types of food-based anticancer agents.
Purported anticancer chemicals not found in freeze-dried
black raspberries are the betalains, which are composed of
reddish to yellowish betacyanins7and minor yellow to or-
ange betacyanins. These pigments are prepared by me-
chanically crushing red beetroots and concentrating the
water-soluble colored juice by spray-drying. The product is
stabilized by the addition of ascorbic acid, citric acid and
a diluent such as dextrin. The final product is sold by the
food industry as red dye E162. Because they have excep-
tionally high radical scavenging activity, the betacyanins
and other betalains are thought to be the most important
anticancer constituents of E162.7–12The only table foods
containing these pigments are red beetroot, cactus pear,
amaranth, pitaya, and Swiss chard.13E162 prepared from
red beetroot contains 0.35–0.5% dry weight of betalains.
It also contains among many other constituents betaine at
*1–1.2% dry weight. Betaine is known to methylate DNA
by the transfer of a methyl group to S-adenosylmethionine
Manuscript received 6 November 2008. Revision accepted 16 June 2009.
Address correspondence to: Gary D. Stoner, Ph.D., Department of Internal Medicine,
Ohio State University Comprehensive Cancer Center, 2001 Polaris Parkway, Columbus,
OH 43240, USA, E-mail: email@example.com
*The first two authors should be considered co-first authors.
JOURNAL OF MEDICINAL FOOD
J Med Food 13 (3) 2010, 733–739
# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
and, ultimately, to the 5 position of cytosine in DNA.14–17
A common feature of tumors is hypomethylation, and
Feinberg et al. and Kisseljova and Kisseljov recently re-
viewed the importance of hypomethylation in carcinogenesis
and suggested that food constituents that antagonize hypo-
methylation should be cancer preventive.18,19Thus, the beta-
ine in E162 may also have chemopreventive activity. Finally,
undefined components may also have anticancer activity.
Kapadia et al. have shown that carcinogen-treated
mice given beetroot extract showed significant tumor in-
hibitory effect.8,9They also reported that the mice that
drank water containing 0.0025% E162 ad libitum had
significantly reduced incidences of tumors in 7,12-
B-promoted and (?)-(E)-4-methyl-2-[(E)-hydroxyamino]-
noylphorbol-13-acetate-promoted skin carcinogenesis and
in N-nitrosodiethylamine-induced phenobarbital-promoted
liver tumors. Because E162 has divergent anticancer con-
stituents, we hypothesized that E162 might also inhibit
NMBA-induced esophageal papilloma development in rats.
Thus, we conducted the present study to determine the po-
tential chemopreventive effects of E162 in the rat esophagus
and to investigate the mechanism(s) of its action.
MATERIALS AND METHODS
Animals, diet, and E162-water treatment
Male F344 rats, 4–5 weeks old (Harlan Sprague-Dawley,
Indianapolis, IN, USA), were housed under standard con-
ditions (20?28C; 50?10% relative humidity; 12-hour
light=dark cycle). The animals were provided AIN-76A
synthetic diet (Dyets, Inc., Bethlehem, PA, USA) and either
deionized water or E162-water. The E162-water was pre-
pared by dissolving 78mg=mL E162 dye powder (single
batch provided by Chr. Hansen, Inc., Milwaukee, WI, USA)
in water. The typical formation (percentage dry weight) of
E162 is as follows: ash, 12.3?0.1; protein, 12.9?0.6;
crude fiber, 0.3?0.1; total sugars, 45.2?0.04; sucrose,
34.0?0.6; pigments (betaalins) 0.5?0.15; and betaine,
1.0?0.3. All animals received their water via bottles that
were exchanged with fresh water or freshly prepared E162-
water three times weekly. The animals were caged in a room
certified balance. As the half-life of betacyanins is known20
to exceed 100 days at 168C and at pH >3, the level of these
constituents in the drinking water remained essentially
constant. Body weight and food and water consumption per
cage were recorded weekly. All experimental protocols
were in accordance with National Institutes of Health
guidelines and approved by the Institutional Animal Care
and Use Committee of The Ohio State University.
The rats were randomized into four experimental groups
of 18 animals each. Groups 1 (control) and 3 (E162) rats
were injected subcutaneously with 0.2mL of 20% dimethyl
sulfoxide (DMSO) in water (NMBA vehicle) three times per
week for 5 weeks. Group 2 and 4 rats were injected sub-
cutaneously with NMBA (0.3mg=kg of body weight) (Ash
Stevens, Inc., Detroit, MI, USA) in 0.2mL of NMBA ve-
hicle three times per week for 5 weeks. Groups 1 and 2
animals were provided regular drinking water, whereas
groups 2 and 4 animals were provided E162-water ad libi-
tum. The rats were sacrificed at 35 weeks by CO2asphyxi-
ation. The esophagus of each rat was opened longitudinally,
and the surface tumors were mapped, counted, and sized.
Lesions greater than 0.5mm in a single dimension were
classified as tumors. One-half of each esophagus and the
remaining tumors were fixed in 10% neutral buffered for-
malin for histopathologic (hematoxylin and eosin and im-
munohistochemistry) evaluations; the remaining tissues
were snap-frozen in liquid nitrogen.
Sections (4mm) of the entire esophagus and all tumors
from five rats per group were stained with antibodies for
antigens that serve as biomarkers for proliferation (Ki-67),
angiogenesis (CD34), inflammation (CD45), and apoptosis
(terminal deoxynucleotidyl transferase dUTP nick end-
labeling [TUNEL]). Ki-67, CD34, and TUNEL were eval-
uated using paraffin sections; CD45 was assessed using
frozen sections. Antigens were retrieved by placing the
slides in a vegetable steamer in target retrieval solution
(Dako, Carpinteria, CA,USA) for 25 minutes. All slideswere
then incubated for 5 minutes with 3% hydrogen peroxide to
block endogenous peroxidase. The slides were then stained
using a Dako Autostainer. Staining conditions forthe primary
antibodies were as follows: Ki-67 (catalog number M7248,
Dako), diluted 1:10, 1 hour at room temperature; CD34
(catalog number AF4117, R&D Systems, Minneapolis, MN,
USA), diluted 1:800, 30 minutes at room temperature; and
CD45 (catalog number 550566, BD Biosciences, Franklin
Lakes, NJ, USA), diluted 1:25, 1 hour at room temperature.
The slides were then incubated with the respective secondary
antibodies as suggested by the manufacturer. The ApopTag
Plus kit (catalog number S7101, Chemicon, Billerica, MA,
USA) was used for the TUNEL staining following the man-
ufacturer’s instructions. All slides were counterstained with
hematoxylin and viewed and photographed at ?200 with a
bright-field microscope mounted with a high-resolution spot
camera. The camera was interfaced with a computer con-
taining a matrix frame grabber and Simple PCI Imaging
Systems image analysis software (Compix Media, Torrance,
CA, USA). Twenty fields per entire esophagus and 30 fields
of all of the tumors per group were analyzed for their staining
intensities. The staining data were compared using analysis
of variance and an unpaired t test (StatView?, SAS Institute,
Cary, NC, USA). A value of P<.05 was considered to be
The animals consumed approximately 15mL of water
daily. There were no significant differences in either water
or food consumption or in body weights among all groups
of animals during the bioassay (data not shown). The le-
sion data are presented in Table 1. There is a statistically
LECHNER ET AL.
significant (P<.01) difference in the numbers of dysplastic
lesions per esophagus between the NMBA group and the
NMBAþE162-water group. In addition, there is a statisti-
cally significant (P<.001) difference in the average number
of tumors between the NMBA group (11.4) and the
NMBAþE162-water group (6.2).
Examples of the biomarker staining for proliferation
(Ki-67), angiogenesis (CD34), inflammation (CD45), and
apoptosis (TUNEL) among the precancerous lesions and
the tumors that developed in the NMBA group and the
NMBAþE162-water group are shown in Figure 1, and the
data developed from analyzing all of the images are sum-
marized in Figure 2. Regarding Ki-67 (Fig. 2a), there was
no difference in the rates of proliferation between the
DMSO and E162 animals within the normal and hyper-
plastic regions of the esophagi. In contrast, the esophagi of
NMBA-treated animals exhibited significantly (P<.005)
elevated and progressively increasing Ki-67 staining within
the hyperplastic, dysplastic, and papillomatous groups. In
addition, the degree of staining in the papillomas was sig-
nificantly greater (P<.005) than in the hyperplastic lesions.
On the other hand, the level of Ki-67 staining in the corre-
sponding lesions of the NMBAþE162 animals was not
significantly different from the normal epithelium of the
DMSO and E162 groups of rats. Thus, the rate of prolifer-
ation in the papillomas that arose in the NMBA animals was
three times that of the control epithelium of the DMSO
animals, whereas the papillomas that developed in the
NMBAþE162 animals proliferated at a rate that was only
slightly greater (30%; P<.04) than that of the normal epi-
thelium of the DMSO animals.
The effect of E162-water on angiogenesis in the lesions,
as assessed by microvessel density, was evaluated by CD34
staining (Fig. 2b). In the group of rats treated with NMBA
only, the microvessel density in the dysplastic lesions and
in the papillomas was 160% and 220% higher (P<.001),
respectively, compared with the normal epithelium of the
DMSO and E162-water groups of animals. On the other
hand, the respective lesions in the NMBAþE162-water
group displayed no significant difference in CD34 expres-
sion compared with the normal epithelium of the DMSO and
E162 groups of animals.
NMBA CD34 staining; (d) NMBAþE162-water CD34 staining; (e) NMBA CD45 staining; (f) NMBAþE162-water CD45 staining; (g) NMBA
TUNEL staining; and (h) NMBAþE162-water TUNEL staining. Color images available online at www.liebertonline.com=jmf.
Immunohistochemistry of regions with papilloma lesions: (a) NMBA Ki-67 staining; (b) NMBAþE162-water Ki-67 staining; (c)
Table 1. Effects of E162-Water on NMBA-Induced Precancerous Lesion Formation in the Esophagi of F344 Rats
Group Normal Epithelial hyperplasia DysplasiaPapillomas
Data are mean?SD values.
Statistically significant relative to the NMBA group as determined by analysis of variance:aP<.01,bP<.001.
RED BEETS FOR ESOPHAGEAL TUMOR PREVENTION
Inflammation was assessed by CD45 staining (Fig. 2c).
The group of rats treated with NMBA only exhibited sig-
nificantly (P<.001) increased levels of inflammatory cells
in their dysplastic and papillomatous lesions compared to
the normal epithelium and hyperplastic regions of their
esophagi. In contrast, the NMBAþE162-water group dis-
played levels of inflammatory cells that were not signifi-
cantly different from the normal epithelium of the DMSO
and E162-water groups. Thus, the influx of inflammatory
cells into the dysplastic and papillomatous lesions of the
NMBA-treated animals was fourfold and sixfold greater,
respectively, than the levels observed in the corresponding
lesions that arose in the NMBAþE162 animals.
Lastly, we evaluated apoptosis (Fig. 2d). The only sig-
nificant level of apoptosis detected by TUNEL staining was
seen in the papillomas that arose in both the NMBA group
and the NMBAþE162 group. The TUNEL staining indi-
cated that the level of apoptosis was roughly twice (P<.05)
as high in the NMBAþE162 group compared with the
NMBA group, suggesting that the E162-water enhanced
Kapadia et al. noted more than 10 years ago that water
containing red beetroot extract (E162 food color) signifi-
cantly inhibited the induction of lung, skin, and liver tumors
in carcinogen-treated mice.8,9Now, by coupling our rat
esophagus results with theirs, it can be concluded that E162-
water has a broad spectrum of chemopreventive potency.
preneoplastic and papillomatous rat esophagus. Data are mean?SD values (n¼5). (b) Effects of E162-water on microvessel density in
preneoplastic and papillomatous rat esophagus as determined by microvessel density reflected by CD34 staining. Data are mean?SD values
(n¼5). (c) Effects of E162-water on inflammation in preneoplastic and papillomatous rat esophagus determined by CD45 (leukocyte common
antigen) staining. Data are mean?SD values (n¼5). (d) Effects of E162-water on apoptotic index in preneoplastic and papillomatous lesions as
measured by TUNEL staining. Data are mean?SD values.
Summary of the immunochemistry analyses. (a) Effects of E162-water on Ki-67 labeling index as a biomarker of proliferation rate in
LECHNER ET AL.
Specifically, it is anticarcinogenic in rats and mice, both
males and females, and for multiple anatomic sites. Fur-
thermore, the anticancer activity of E162 is manifested at a
low concentration in drinking water (0.0025% and 0.0078%
for mice and rats, respectively) and without overt toxicity
to the animals. In contrast, prevention by freeze-dried black
raspberries of NMBA-induced tumors in rats requires that
the animals consume a daily diet consisting of 5% freeze-
dried berries, or 0.4–4g=kg ellagic acid, or 0.1mmol=kg
phenylethyl isothiocyanate.21Thus, although E162 is not
a pure chemical, as is ellagic acid and phenylethyl iso-
thiocyanate, it is evident that it is a mixture of constituents
that have potent chemopreventive activity. Numerous
investigators have credited betacyanins and other betalains
as having the anticancer activity in E162 preparations.7–13
As E162 is reported by the manufacturer to contain 0.35–
0.5% betacyanins and the rats consumed *1.1mg of E162
daily, it can be calculated that the animals received a steady-
state daily dose of *5mg (*20mg=kg) of betacyanins.
Thus, if betacyanins are the primary active constituent of
E162, it can be inferred that they have very potent cancer
Betacalins are antioxidants. Stintzing et al. reported
that betacyanins are twofold more active as antioxidants
than anthocyanins,22and Kanner et al. and Gliszczyn ´ska-
Swigło et al. noted that they were >10-fold more effective
than a-tocopherol in inhibiting peroxidation.10,11Thus, they
are expected to antagonize inflammation. Inflammation-
causing neutrophils and macrophages are frequently re-
cruited into tumors and produce exogenous reactive oxygen
species.23–26In keeping with the reports that betacyanins are
antioxidants, we compared the level of inflammatory lym-
phocytes in the lesions that developed in NMBA-treated rats
that either did or did not drink E162-water using antibodies
to the leukocyte common antigen CD45.27Our data show
that the tumors that developed in the animals that drank
E162-water had significantly (P<.001) less lymphocyte
infiltration, compared against the NMBA-alone animals.
Furthermore, these experiments showed that the level of
lymphocyte infiltration was not significantly different from
the normal esophageal tissue of the DMSO (control) group.
These results suggest that the betacyanins in E162 antago-
nize lymphocyte infiltration and reduce the level of in-
flammation. They also suggest that one of the mechanisms
of E162 chemoprevention is reducing inflammation.
The xenotransplant studies of Zou et al. suggested that
there was reduced angiogenesis in the tumors that developed
in nude mice that received the cactus pear preparation of
E162.28Accordingly, we quantified microvessel densities in
precancerous esophageal lesions and tumors in animals that
drank E162-water, using the CD34 antibody.29The data
showed that the precancerous lesions and tumors that arose
in the animals that drank E162-water had a microvessel
density that was 40% lower than that in the esophagi of
NMBA-alone animals.Thus, our results suggestthat another
cancer preventing activity of E162 is to antagonize angio-
genesis. As angiogenesis is promoted by reactive oxygen
species,30some of the chemopreventive activity may be
attributed to the antioxidant action of the betacyanins and
Other in vitro observations suggest that betacyanins and
other betalains depress proliferation. Sreekanth et al.
showed that E162 caused a dose- and time-dependent
decrease in cellular proliferation.31Therefore, we quantified
the levels of proliferation in precancerous lesions and
in esophageal tumors, using Ki-67 staining. Our data
show that the level of proliferation in these lesions in the
NMBAþE162 rats remains at control levels, regardless of
grade of pathology. In contrast, Ki-67 staining was signifi-
cantly (P<.001) elevated in the hyperplastic, dysplastic,
and papillomatous lesions that arose in the NMBA-only
rats. Again, it can be hypothesized that the betacyanins and
other betalains antagonized proliferation because their an-
tioxidative activity reduced the level of reactive oxygen
species to levels that were too low to stimulate proliferation
via inappropriate signal transduction.32Betacyanins and
other betalains seemingly have also chemotherapeutic ac-
tivities. Zou et al. showed that their E162-like extract of
cactus pear was effective in slowing the growth of tumors
after injection of SKOV3 human ovarian cancer cells in
nude athymic mice.28Their observation matches our Ki-67
data showing that the papillomas that developed in the
NMBAþE162 animals proliferated more slowly compared
to those that were found in the NMBA-alone rats. Alter-
natively, the reduced proliferation could be due to mecha-
nisms that augment apoptosis.33Accordingly, we evaluated
apoptosis levels using the standard TUNEL assay. We found
that the lesions in the esophagi from the animals that drank
E162-water had relatively (P<.05) higher levels of apo-
ptosis. Again, the antioxidant activity of the betacyanins in
E162 may be responsible for this observation, as excessive
reactive oxygen species can antagonize TP53-caused apo-
ptosis by oxidative inactivation of the protein. As a conse-
quence the lesion=tumor exhibits less apoptosis.34
Future purification studies are needed to affirm that beta-
cyanins and other betalains are the primary chemopreventive
constituents in E162. Another candidate is betaine.14–16This
C1 donor may constitute as much as 1.5% of the dry weight
diet can develop hypomethylation and cancers.35Thus, the
betanin in E162 may antagonize cancer development by
augmenting S-adenosylmethionine levels,36,37which, in
turn, antagonize the development of hypomethylation.18,19
In addition, betaine is anti-inflammatory.38Future studies
will look into this possible anti-hypomethylation or anti-
inflammation role of betaine in the mechanism of E162
In conclusion, the goal of chemoprevention is to prevent
or greatly slow the processes that lead to excessive repli-
cation of damaged cells that have arisen from many years of
carcinogen insults. Another characteristic is that the com-
pound must have little or no long-term toxicity. It must also
be sufficiently potent to provide protection at low doses,
easy to administer, and inexpensive. Our results with E162
using a rat esophageal squamous cell carcinoma model show
that it can antagonize tumorigenesis. It is also known to be
RED BEETS FOR ESOPHAGEAL TUMOR PREVENTION
effective as a chemopreventive against mouse skin, lung,
and liver cancer. The antagonism requires small amounts
(<50mg=day for a 70-kg human) incorporated into drinking
water, it has no known toxicity,39and it is inexpensive.
Thus, E162 adequately satisfies the required characteristics
to be a chemopreventive food product. Finally, as the active
constituents in E162 (betalains and betaine) are different
from those in freeze-dried black raspberries, future studies
will determine if treatment of rats with both E162 and
freeze-dried black raspberries will cause a further reduction
in NMBA-induced esophageal tumors in rats.
This research was supported in part by American Institute
for Cancer Research grant 07A104 (to J.F.L.) and National
Institutes of Health grant CA103189 (to G.D.S.).
AUTHOR DISCLOSURE STATEMENT
All authorsdeclare no compelling financial interestsexist.
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