Carcinogenesis vol.19 no.8 pp.
1445–1449, 1998
Studies of iron deposits, inducible nitric oxide synthase and
nitrotyrosine in a rat model for esophageal adenocarcinoma
Susan R.Goldstein1, Guang-yu Yang1, Xiaoxin Chen1,
Stephen K.Curtis2 and Chung S.Yang1,3
1Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State
University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-
2
8020 and Laboratory Animal Services, Rutgers, The State University of
New Jersey, Nelson Biological Laboratory, 604 Allison Road, Piscataway,
NJ 08854-8082, USA
3To
whom correspondence should be addressed
Email: csyang@rci.rutgers.edu
A rat model was developed recently in our laboratory to
study the pathogenesis of Barrett’s esophagus (BE) and its
progression to esophageal adenocarcinoma (EAC). Eight-
week-old male Sprague–Dawley rats underwent esophago-
duodenal anastomosis (EDA) to produce gastric and
duodenal reflux in their distal esophagi. The rats were
given iron dextran (50 mg of Fe/kg, i.p.) starting 2 weeks
after surgery and this was continued once a month. BE
was observed as early as week 3 and the incidence of BE
and EAC increased with time: 58 and 17% at week 23; 91
and 73% at week 31. There was a progression in epithelial
cell proliferation and inflammation from mild to severe in
the distal one-third of the esophagus. Iron deposition in
the esophagus also increased with time. Iron deposits in
the stromal tissue adjacent to the epithelium in the distal
one-third of the esophagus were associated with areas of
severe inflammation. Immunohistochemical analysis
showed positive inducible nitric oxide synthase (iNOS)
expression in the stromal macrophages directly beneath
the epithelium in the distal one-third of the esophagus in
36, 83 and 100% of the rats at weeks 17, 23 and 31,
respectively. A significant increasing linear trend (P J
0.001) was seen in nitrotyrosine immunostaining (number
of positive cells/high power field) in the distal esophagus.
Strong positive nitrotyrosine staining was seen in the
macrophages and weaker positive staining was seen in the
adjacent epithelium starting at week 17. Furthermore, iron
supplemented rats killed at week 31 had significantly higher
(P < 0.05) levels of inflammation, cell proliferation, iNOS
and nitrotyrosine as well as more tumors in their distal
esophagi than did rats that received no iron supplement.
These results suggest that iron supplementation enhanced
inflammation and the production of reactive oxygen and
nitrogen species in the esophageal epithelium. These pro-
cesses could contribute to the formation of BE and its
progression to EAC.
Introduction
Esophageal adenocarcinoma (EAC*) is a disease with a rapidly
increasing incidence and an extremely poor prognosis (1–4).
*Abbreviations: ABC, avidin–biotin peroxidase complex; BCH, basal cell
hyperplasia; BE, Barrett’s esophagus; DAB, diaminobenzidine; EAC, eso-
phageal adenocarcinoma(s); EDA, esophagoduodenal anastomosis; H&E,
hematoxylin and eosin; h.p.f., high power field; iNOS, inducible nitric oxide
synthase; NO, nitric oxide; PBS, phosphate buffered saline; SCC, squamous
cell carcinoma; TUF, target unmasking fluid.
© Oxford University Press
Intestinal-type Barrett’s esophagus (BE), a condition in which
the normal stratified squamous epithelium is replaced by
metaplastic columnar epithelium containing goblet cells, is a
frequent precursor lesion of EAC (5,6). Patients with BE have
a 30–125-fold increased risk over the general population of
developing EAC (7–9). It is widely accepted that BE is
an acquired condition caused by chronic gastroesophageal
reflux (3,7,10).
Esophagoduodenal anastomosis (EDA, surgery to connect
the esophagus to the duodenum, also known as duodenoeso-
phagostomy) produces gastric and duodenal reflux into the
esophagus, but produces EAC in rats at very low rates, if at all
(11–13). Treatment of the operated animals with a carcinogen
markedly enhances cancer development (45–80%). Most of
the esophageal tumors had mixed properties of squamous cell
carcinomas (SCC) and EAC, with cells producing keratin in
one area and mucin in another. Only a small percentage of
tumors were pure well-differentiated EAC.
We reproduced these results with EDA and treatment with
a carcinogen, methyl-N-amyl nitrosamine or N9-nitrosonornico-
tine (14). We also found that the operated animals were
severely anemic several months after surgery and that this
condition was responsive to iron treatment (14,15). In a second
experiment, iron dextran (50 mg of Fe/kg, i.p., monthly) was
used to prevent the development of anemia in the operated
rats. This treatment, without a carcinogen, produced a high
rate of intestinal BE (91%) and EAC (73%) in 31 weeks (14).
The morphology of rat BE and EAC resembled that of human
BE and EAC in many respects, and all of the EAC was pure
well-differentiated mucinous EAC.
Chronic gastroesophageal reflux is believed to damage the
normal stratified squamous epithelia and produces a compen-
satory hyperproliferative response in the human and rat eso-
phagi (12,16,17). Induced cell proliferation has been reported
to play a key role in carcinogenesis (18–20). Cell proliferation
facilitates the conversion of DNA damage to mutations, leading
to the activation of oncogenes or inactivation of tumor sup-
pressor genes and a dysregulation of proliferation controls.
Damage to the normal human or rat esophageal squamous
epithelium by chronic gastroduodenal reflux also produces an
inflammatory response in the distal esophagus (12,16,17).
Chronic inflammation has long been recognized as a risk factor
for a variety of human cancers (21,22). Reactive oxygen
species and reactive nitrogen species produced by inflammatory
phagocytes are important components of inflammation and
have also been shown to cause significant tissue injury. It is
possible that these reactive species contribute to all stages of
carcinogenesis: initiation, promotion and progression (23,24).
Iron is known to be the most effective metal ion in catalyzing
the production of free radicals (25,26). Epidemiological studies
have shown that increased body stores of iron are associated
with an increased risk of cancer (27–29). An increased produc-
tion of tumors was also observed in animal models (30–32).
We hypothesize that iron supplementation to the EDA rats
1445
S.R.Goldstein et al.

enhanced esophageal inflammation and the production of

reactive oxygen (nitrogen) species. These species could damage Table I. Time-dependent progression of proliferation, inflammation, and
epithelial cells and play a major role in adenocarcinogenesis iron deposition in the esophagus of rats that have undergone EDA and
received iron supplementationa
in the rat. To test this hypothesis we characterized cell
proliferation and inflammation, investigated iron deposition, Week n Proliferationb Inflammationb Iron depositsc
and examined evidence of nitric oxide production in the
esophagi of rats that had undergone EDA and received iron 3 5 1.4 6 0.55 1.0 6 0 0.2 6 0.45d
5 6 2.2 6 0.41 1.8 6 0.75 2.2 6 0.98
or no iron supplement. 9 5 2.0 6 0 1.2 6 0.45 2.0 6 0.71
17 13 2.3 6 0.63 2.4 6 0.65 2.6 6 0.51
Materials and methods 23 12 2.5 6 0.91 2.1 6 0.79 2.6 6 0.5
Materials 31 11 2.7 6 0.47 2.9 6 0.30 2.8 6 0.41
Iron dextran was purchased from Henry Schein (Port Washington, NY). The aIron
chemicals hematoxylin, eosin, potassium ferrocyanide and neutral red were dextran (50 mg Fe/kg) was administered i.p. monthly starting 2 weeks
purchased from Sigma (St Louis, MO). Target unmasking fluid (TUF) after surgery.
bGraded on a scale of 0–3 using morphological changes (0, normal; 1, mild;
was purchased from Pharmingen (San Diego, CA). Peroxidase avidin and
biotinylated horseradish peroxidase macromolecular complex (ABC) kits were 2, moderate; 3, severe). Values are means 6 SD. There was a significant
purchased from Vector Laboratories (Burlingame, CA). Anti-NOS2, a rabbit increasing linear trend for proliferation (P 5 0.0003) and for inflammation
polyclonal antibody, was purchased from Santa Cruz Biotechnology (Santa (P 5 0.0001).
cGraded on a scale of 0–3: 0, no iron; 1, ,5 scattered positive cells/h.p.f.;
Cruz, CA). Anti-nitrotyrosine, a rabbit polyclonal antibody, was purchased
from Upstate Biotechnology (Lake Placid, NY). 2, 5–30 positive cells/h.p.f.); 3, .30 positive cells/h.p.f. Values are means
6 SD. There was a significant increasing linear trend (P 5 0.0001) for iron
Animals and treatment procedures deposition.
Six-week old male Sprague–Dawley rats from Taconic Farms (Germantown, dOnly one animal was positive for iron. It had two positive-stained cells in
NY) were housed at one per cage, given commercial rat chow and water ad the distal esophagus.
libitum, and maintained on a 12 h light–dark cycle. They were allowed to
acclimate for 2 weeks prior to surgery. Solid food was withdrawn 24 h to phosphate buffered saline (PBS). Target unmasking fluid was used to
prior to surgery. EDA was performed according to the procedure described unmask the antigen. The endogenous peroxidase activity was quenched with
previously (12,14). This procedure was approved by the Animal Care and hydrogen peroxide. Normal sera (10% normal goat serum) was applied to
Facilities Committee, Rutgers University (protocol no. 94-017). Postopera- minimize non-specific binding. A 1 h incubation with the first antibody (5
tively, the animals were given water after 2 h and rat chow the following day. µg/ml for iNOS and 2 µg/ml for nitrotyrosine) was followed by the application
The surgical animals were given iron dextran (50 mg Fe/kg, i.p.), starting 2 of the secondary antibody conjugated to biotin. An avidin–biotin peroxidase
weeks after surgery (i.e. week 3) and continuing once a month for the duration complex (ABC) was then applied, and the staining was visualized with
of the experiment to prevent anemia. The animals were weighed weekly. diaminobenzidine (DAB). The sections were counterstained with Mayer’s
Tissue preparation hematoxylin, washed, and then dehydrated using a gradient of ethanol to
The rats were anesthetized with CO2, 2 ml of blood were taken by cardiac xylene. The slides were cover-slipped using permount. The percentage of
puncture, and then they were killed by CO2 asphyxiation. The stomach, the cases that stained positive for iNOS at each time point was determined. For
esophagus and part of the duodenum were removed, opened longitudinally, nitrotyrosine evaluation, one slide per rat was stained using the nitrotyrosine
photographed and examined for gross abnormalities. The tissue was fixed in antibody. Four slides were chosen at random from each group. For each slide,
10% buffered formalin for 24 h and then transferred to 80% ethanol. The the number of cells staining positive for nitrotyrosine/h.p.f. were counted
esophagus was cut 2–3 mm above the blue prolene sutures to eliminate the manually for three fields at the lower esophagus and three fields at the middle
duodenal mucosa that may have puckered during the healing process. A Swiss esophagus. Thus, a total of 12 measurements each for the lower and middle
roll of the esophagus was made, processed and embedded in paraffin. Sections esophagus were made per group. The data were evaluated statistically using
(5 µm) of tissue were mounted onto glass slides and used for hematoxylin a linear regression model.
and eosin (H&E) staining and immunohistochemistry.
Morphological analyses Results
Epithelial cell proliferation on the distal third of the esophagus was graded Cell proliferation and inflammation
based on morphology, with a scale of 0–3, using the H&E stained tissue
slides. The normal stratified squamous epithelium with 1–3 basal cell layers Cell proliferation and inflammation in the distal esophagus
was graded as 0. Basal cell hyperplasia was graded as 1 for mild, 2 for were analyzed as a function of time. Basal cell hyperplasia
moderate and 3 for severe when the number of proliferating basal cell layers (BCH) and inflammation were seen at all time points after
occupied over one-third, one-third to two-thirds and over two-thirds of the surgery. There was a significant increasing linear trend for
total epithelial thickness, respectively. Inflammation was also graded based
on morphology using the H&E stained slides, with the following criteria: 0, both cell proliferation (P 5 0.0003) and inflammation (P 5
normal (no inflammatory cells); 1, mild (a few scattered inflammatory cells/ 0.0001) (Table I). Overall, BCH was graded as mild at week
high power field, 3525, in the stromal tissue); 2, moderate (moderate density 3, mild to moderate at week 5, moderate at weeks 9 and 17,
of lymphocytes and macrophages); and 3, severe (high density of lymphocytes and moderate to severe at weeks 23 and 31. The hyperplastic
and macrophages with an enlargement of capillaries in the stromal tissues).
The data were evaluated statistically using a linear regression model.
epithelium covered the bottom half of the esophagus at week
Iron deposition in the esophagus
3, and the bottom three-quarters at the later time points. Based
Iron histochemical staining was done using the Prussian blue test for ferric
on the density of inflammatory cells and the ingrowth of
iron. This test involves the following procedure: the tissue sections were capillaries in the submucosal stroma tissues, inflammation was
dewaxed with xylene and then hydrated with graded ethanol to water. The graded as mild to moderate at 3, 5, 9 and 17 weeks, moderate
slides were then immersed in ferrocyanide solution (containing 2% potassium to severe at week 23, and severe at week 31. The most severe
ferrocyanide and 2% aqueous hydrochloric acid), washed, and then coun- inflammation and epithelial damage were observed in the
terstained with 1% neutral red. After washing, the tissue sections were
dehydrated in graded ethanol, cleared in xylene, and then mounted using esophagoduodenal junction, characterized by epithelial erosion
permount. The iron stained bright blue; the rest of the tissue stained red. The and chronic ulceration with numerous inflammatory cells
slides were graded for iron: 0, no iron staining; 1, ,5 scattered positive cells/ (lymphocytes, eosinophils and macrophages), dilation of ven-
h.p.f. in the stromal or muscle tissue; 2, 5–30 positive cells/h.p.f.; 3, .30 ules and proliferation of the capillaries.
positive cells/h.p.f. The data were evaluated using a linear regression model.
Inducible nitric oxide synthase (iNOS) and nitrotyrosine immunohistochemistry
Iron deposition in the esophagus
The basic immunohistochemistry procedure (33) was as follows: the tissue A significant increasing linear trend (P 5 0.0001) was seen
sections were first dewaxed in xylene, and hydrated in a gradient of ethanol in iron deposition in the distal esophagus (Table I). Positive
1446
 Iron deposits, iNOS and nitrotyrosine in esophageal adenocarcinogenesis

Fig. 2. Micrographs showing iNOS immunohistochemical staining in the

distal esophagi of rats that had undergone EDA and received iron
supplementation. BCH at week 5, no iNOS staining (3500); BCH at week
17, with iNOS staining (3500); BE at week 31, with iNOS staining
Fig. 1. Micrographs showing iron accumulation, at week 31, in the stromal (3500).
tissue of a rat that had undergone EDA and received monthly iron
supplementation (50 mg Fe/kg, i.p.). Normal, upper one-third of the
esophagus (3500); BCH, iron accumulation next to moderate BCH (3500);
BE, iron accumulation next to BE (3500); EAC, iron accumulation next to
EAC (3500).

Table II. Time-dependent expression of iNOS in the esophagus of rats that

had undergone EDA and iron supplementationa

Week n iNOS expression (%)b

3 5 0
5 6 0
9 5 0
17 13 36
23 12 83
31 11 100
aIrondextran (50 mg of Fe/kg) was administered i.p. monthly starting 2 Fig. 3. Micrographs showing nitrotyrosine immunohistochemical staining, at
weeks after surgery. week 31, in the distal esophagus of a rat that had undergone EDA and
bPercent of animals showing positive immunochemical staining.
received iron supplementation. Normal, upper one-third of the esophagus
(3500); BCH, strong staining in the stromal macrophages, weaker staining
in BCH (3500); BE (3500), strong staining in the stromal macrophages,
iron staining was seen in the muscle layer as well as in the weaker staining in BE; BE (31000), close-up of previous frame.
submucosa (or stromal tissue) adjacent to the epithelia in the
distal one-third of the esophagus in all of the iron-injected
animals (Figure 1). Positive iron staining was not uniformly
distributed in the esophagus; it was associated with severe
inflammation in areas with or without erosion of the epithelial
layers. Positive iron staining was seen along the edges of the
tumors and not in the center of the cancer nests. The operated
animals that were not injected with iron and the non-operated
animals did not have positive iron staining in their esophagi.
iNOS and nitrotyrosine immunohistochemistry
iNOS expression was not observed at weeks 3, 5 and 9, but
there was an increase in iNOS expression at later time points.
At week 17, 36% of the animals had iNOS expression in the
distal esophagus. This percentage increased to 83% at week Fig. 4. Number of nitrotyrosine-positive cells determined by
23 and 100% at week 31 (Table II, Figure 2). All of the immunohistochemistry in the stromal macrophages per high power field in
positive iNOS staining was found in the stromal macrophages the middle and lower esophagi of rats that had undergone EDA: j, lower
directly beneath the epithelium in the distal one-third to two- esophagus; d, middle esophagus.
thirds of the esophagus. No positive iNOS staining was seen
in the upper one-third of the esophagi of the operated animals, the lower one-third to two-thirds of the esophagi of all the
or in the non-operated animals. operated animals; most of the positive staining was seen in
There was a significant increasing linear trend (P 5 0.0001) the lower esophagus and a lesser amount was seen in the
in nitrotyrosine formation (number of positive cells/h.p.f.) in middle esophagus. More importantly, weaker positive staining
the lower esophagus, but not in the middle esophagus (Figures was seen in the basal and parabasal cells in BCH and in
3 and 4). Strong positive nitrotyrosine staining was found in Barrett’s epithelium at weeks 17, 23 and 31 (Figure 4). No
the stromal macrophages directly beneath the epithelium in positive nitrotyrosine staining was seen in the upper esophagi
1447
S.R.Goldstein et al.
Table III. Comparison of EDA rats that had received iron supplementation
with those withouta
n Inflammationb Proliferationb Nitrotyrosineb iNOSc Tumorc
Iron 11 2.9 6 0.3* 2.7 6 0.5* 76.8 6 11.1* 100%* 73%*
No iron 4 2.0 6 0.8 1.3 6 0.5 15.5 6 5.3 0 0
aIron dextran (50 mg Fe/kg) was administered i.p. monthly starting 2 weeks
after surgery and this group of rats was killed at week 31. The ‘no iron’
group did not receive iron supplementation and was from a previous
experiment, that were killed 26 weeks after surgery. Means 6 SD values
and percent of animals with iNOS staining and tumors are shown.
bThere was a significant difference in proliferation (P 5 0.001)
inflammation (P 5 0.003) and nitrotyrosine staining between the two
groups using the unpaired Student’s t-test.
cThere was a significant difference (P , 0.05) in the percentage of rats with
iNOS staining and with tumors between the two groups, using the chi-
square test for a small sample size.
*Significantly higher (P , 0.05).
of the operated animals or in the esophagi of the non-
operated animals.
Comparison of iron-treated rats with non-iron treated rats
In order to study the effect of iron supplementation on EDA
rats, rats with iron supplementation were compared with those
without (Table III). The supplemented group (killed at week
31) had significantly higher (P , 0.05) levels of inflammation,
cell proliferation, iNOS expression and nitrotyrosine, as well
as more tumors than the EDA rats, which did not receive
iron supplementation (killed 26 weeks after surgery). Iron
supplemented rats killed at week 23 also had significantly
higher (P , 0.05) levels of cell proliferation, iNOS and
nitrotyrosine than rats in the latter group (data not shown).
Discussion
Iron supplementation to EDA rats produced a high incidence
of BE (91%) and EAC (73%) in week 31 with the EDA model
(14), whereas EDA without iron supplementation produced a
low incidence (0–18%) of EAC (11–13). We propose that
iron enhances inflammation-induced oxidative stress, which
damages the esophageal epithelium and contributes to the
development of BE and EAC. This hypothesis is supported by
the comparison of the iron-treated EDA rats with EDA rats
that received no iron supplementation. The iron-treated rats
had significantly higher levels of iron deposition, inflammation,
cell proliferation, iNOS and nitrotyrosine, as well as more
tumors (Table III). We have characterized BCH and inflamma-
tion in the development of BE and EAC with the rat EDA
model. BCH and inflammation increased from mild, to moder-
ate, to severe over the course of the experiment. There was a
significant increasing linear trend in both cell proliferation and
inflammation. Iron deposition in the stromal tissue adjacent to
the epithelium in the distal one-third of the esophagus, increased
with time and was associated with areas of severe inflammation.
Since the iron was administered i.p., we believe that the iron
was picked up by the macrophages and then deposited in the
esophageal stromal tissue. These iron deposits could enhance
inflammation as well as the production of reactive oxygen
radicals.
We saw positive iNOS immunohistochemical staining in the
stromal macrophages in the distal one-third of the esophagus,
starting at week 17 after surgery (36%) and with increased
frequencies at week 23 (83%) and week 31 (100%). However,
1448
positive nitrotyrosine immunohistochemical staining was seen
in the stromal macrophages in the distal one-third of the
esophagus at all time points after surgery, starting at 3 weeks.
A significant increasing linear trend was seen in nitrotyrosine
expression (number of positive cells/h.p.f.) in the stromal
macrophages. We expected the expression of iNOS and nitro-
tyrosine to match, because nitric oxide is required for the
synthesis of peroxynitrite, which reacts with tyrosine to form
nitrotyrosine (34). One explanation for this disparity might be
that the nitrotyrosine immunochemical staining was more
sensitive than that for iNOS; the iNOS expression was not
visualized until high protein levels were reached. Nitric oxide
might have been produced continuously at low levels from
weeks 3 to 9, which might have been at levels high enough
to generate nitrotyrosine accumulation.
A significant finding of our present work was the detection
of positive nitrotyrosine immunostaining in the epithelial cells,
(both BE and BCH) in the distal one-third of the esophagus,
starting at 17 weeks after surgery. Although the epithelial
staining was weaker than that seen in the stromal macrophages,
this result links nitric oxide production in the stromal macro-
phages with oxidative damage in the esophageal epithelial
tissue, which may be important in carcinogenesis.
Although nitric oxide has been shown to have many normal
physiological functions, excess nitric oxide production can
have many potentially damaging effects on epithelial cells,
including (i) interaction with mitochondrial iron–sulfur proteins
and interfering with their functions; (ii) nitrosation of amines
to produce nitrosamines, many of which are potent carcinogens;
(iii) reaction with primary amine groups in DNA to result in
deamination and mutation; (iv) inhibition of the repair of O6-
methylguanine in DNA by forming an S–NO adduct with O6-
methylguanine–DNA methyltransferase; and (v) reaction with
superoxide anion to form the relatively long-lived strong
oxidant, peroxynitrite, which can react with thiol groups and
tyrosyl residues in proteins (21–23,35,36). Because tyrosine
nitration is an irreversible reaction, 3-nitrotyrosine accumula-
tion is considered to be a cumulative index of peroxynitrite
production (35,37). Tyrosine nitration has been reported to
interfere with phosphorylation by tyrosine kinases, which in
turn may affect the ability of the protein to participate in
important signaling pathways (23,35,37). Nitrotyrosine produc-
tion has been associated with the induction of oxidative stress
in human and animal models of diseases, including human
rheumatoid arthritis (38) and atherosclerosis (34), human
Opisthorchis viverrini infection associated with cholangio-
carcinomas (22), and animals chronically infected with hepatitis
virus (21).
In previous work, we demonstrated that iron supplementation
to rats given EDA enhanced the incidence of EAC, and this
histopathogenic process is similar to that reported for human
BE and EAC (14). In the present work, we observed that iron
supplementation caused the deposition of iron and the induction
of iNOS in the stromal macrophages of the distal esophagus.
In human BE and EAC samples, similar patterns of iron
deposition (G.-Y.Yang, N.Altork and C.S.Yang, unpublished
results) and iNOS induction (39) have also been observed.
Even though these events were detected only in inflammatory
cells in the stroma, the reactive oxygen (nitrogen) species
produced could elicit damage to micromolecules in epithelial
cells and Barrett’s cells as indicated by nitrotyrosine formation
in these cells. These could be important events in the formation

of BE as well as its progression to EAC. More quantitative
data, however, are needed to further substantiate this point.
The present surgical model induces reflux of duodenal and
gastric juice into the esophagus and produces BE and EAC.
Although the pathogenic processes resemble those seen in
humans, this is a rather drastic model, which produces features
not shared by patients with BE. For example, the rats given
EDA have a lower nutritional status in terms of iron, vitamin
A and vitamin E because of malabsorption (X.X.Chen,
G.-Y.Yang and C.S.Yang, unpublished results). A key feature
of our model is iron supplementation. It is not known whether
iron overload is a risk factor in human esophageal adenocarcin-
ogenesis. The fact that the incidence of BE and EAC is more
prevalent in North American and European males than females
(a ratio of 8:1) (1) is consistent with the iron overload
hypothesis. The consumption of red meat in these areas is
high and males have a higher possibility of having iron
overload because they do not have a normal route for iron
elimination. The contribution of iron overload to human
esophageal adenocarcinogenesis remains to be investigated.
Acknowledgements
This study was supported by NIH grant CA75683 and facilities from Center
grant ES05022 and CCSG grant CA72720.
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Received on November 21, 1997; revised on April 1, 1998; accepted on April
28, 1998
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