[CANCER RESEARCH 59, 4823–4828, October 1, 1999]

High-Salt Diet Induces Gastric Epithelial Hyperplasia and Parietal Cell Loss, andEnhancesHelicobacter pyloriColonization in C57BL/6 Mice1

James G. Fox,2 Charles A. Dangler, Nancy S. Taylor, Amy King, Theodore J. Koh, and Timothy C. WangDivision of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 [J. G. F., C. A. D., N. S. T., A. K.], and Gastrointestinal Unit,Massachusetts General Hospital, Boston, Massachusetts 02144 [T. J. K., T. C. W.]

ABSTRACT

A high-salt diet in humans and experimental animals is known to causegastritis, has been associated with a high risk of atrophic gastritis, and isconsidered a gastric tumor promoter. In laboratory rodents, salt is knownto cause gastritis, and when coadministered, it promotes the carcinogeniceffects of known gastric carcinogens. BecauseHelicobacter pylorihas beenassociated with a progression from gastritis to gastric cancer, we designeda study to determine whether excessive dietary NaCl would have an effecton colonization and gastritis in the mouse model ofH. pylori infection.Seventy-two, 8-week-old female C57BL/6 mice were infected withH.pylori strain Sydney, and 36 control mice were dosed with vehicle only.One-half of the infected and control mice were fed a high-salt diet (7.5%versus 0.25%) for 2 weeks prior to dosing and throughout the entireexperiment. Twelve infected and 6 control animals from the high-salt andnormal diet groups were euthanized at 4, 8, and 16 weeks. At 8 and 16weeks postinfection (WPI), the colony-forming units per gram of tissuewere significantly higher (P < 0.05) in the corpus and antrum of animalsin the high-salt diet group compared with those on the normal diet.Quantitative urease was significantly higher (P < 0.05) at 4 and 8 WPI inthe corpus and antrum of animals on the high-salt diet when comparedwith controls. At 16 WPI, mice in both the normal and the high-salt dietgroups developed moderate to marked atrophic gastritis of the corpus inresponse toH. pylori infection. However, the gastric pits of the corpusmucosa in mice on the high-salt diet were elongated and colonized byH.pylori more frequently than those in mice on the normal diet. The high-saltdiet was also associated with a significant increase in proliferation in theproximal corpus and antrum and a multifocal reduction in parietal cellnumbers in the proximal corpus, resulting in the elongation of gastric pits.We conclude that excessive NaCl intake enhancesH. pylori colonization inmice and in humans and that chronic salt intake may exacerbate gastritisby increasing H. pylori colonization. Furthermore, elevated salt intakemay potentiate H. pylori-associated carcinogenesis by inducing prolifera-tion, pit cell hyperplasia, and glandular atrophy.

INTRODUCTION

Salt and its role in the development of gastric cancer has beendebated for decades (1–4). Evidence of its deleterious effects is basedon epidemiological studies, biochemical analyses, andin vivo exper-imentation (5, 6). Studies have associated an increased risk of gastriccancer with the ingestion of preserved, often salty food (7) or apreference for salty foods (2, 8). This is further supported by theanalysis of study data that incorporate the measurement of salt con-sumption (9–12). Given the late onset of gastric cancer in humanpopulations, it is probable that both tumor initiators as well as tumorpromoters play an important role in gastric carcinogenesis. In exper-

iments with rats dosed with MNNG,3 NaCl has been shown to have apromoting effect on induction of gastric adenocarcinoma when salt isgiven intragastrically weekly or in the diet during 12–20 weeks ofexposure to MNNG in drinking water (13–15). It was initially estab-lished that ingestion of salt by rats led to chronic injury to rat gastricsurface epithelium, followed by the proliferation of gastric epithelium(16–19). Studies also showed that 80% of rats given salt in the dietand MNNG in drinking water developed adenocarcinomas in theantrum but not in the corpus (14). In addition, ACI rats given a singleinitiation dose of MNNG [5 g/L MNNG in water, 0.25 ml/10 g bodyweight] by intubation and fed a 10% NaCl diet had a significantlyincreased number of tumors in both forestomach and glandular stom-ach after being on the salt diet for 1 year (20). Thus, studies in ratssuggest that a variety of factors probably contribute to the cocarcino-genic effect of salt in gastric cancer.

Mice have also been used on a limited basis in studies addressinggastric damage due to salt intake. Mice fed a rice diet containinghighly salted food developed acute gastric mucosal damage (21). Inlater studies, Swiss/ICR mice fed salted [10% NaCl (w/w)] rice dietsfor 3–12 months developed hypertrophy of the forestomach andatrophy (considered a marker of premalignancy in humans) of theglandular stomach (22). The authors emphasized that a reduction inparietal cell mass accounted for the atrophy observed in the corpus ofthe mice (22). Overall, these studies in rodents support the hypothesisthat salt can contribute to atrophy and function as a cocarcinogen.However, the precise role of these models in relationship toHelico-bacter-associated gastric disease has not been addressed.

It is now known that there is a strong association betweenHelico-bacter pylori and chronic atrophic gastritis (23). This suggests thatHelicobacter infection may also play a role in the development ofgastric cancer. Evidence supporting this observation includes numer-ous studies documenting thatH. pylori infection causes chronicatrophic gastritis. In some cases (particularly in certain populations),chronic gastritis progresses to atrophy, intestinal metaplasia, anddysplasia, features that are consistent with Correa’s model of progres-sion to gastric cancer (24, 25). This hypothesis has been supported byan increasing number of epidemiological studies reported in theliterature beginning in the late 1980s to the present, of which almostall have concluded thatH. pylori is the missing environmental factorin the multifactorial pathogenesis of gastric cancer (24–26). Impor-tantly, mouse models ofH. felis (27, 28) andH. pylori (29) haveshown that chronicHelicobacterinfection of inbred mouse strains canlead to atrophy, metaplasia, and preneoplastic lesions. However, al-thoughH. pylori infection is now accepted as the preeminent envi-ronmental factor in gastric cancer, the possible interactions betweenH. pylori and dietary factors such as salt have not been studied.

To investigate these possible interactions, we designed an experi-ment in which mice infected withH. pylori were fed a high-salt dietto ascertain whether both gastric infection and elevated dietary saltincreased the severity of gastric lesions and affected levels ofH.pylori colonization.

Received 2/23/99; accepted 8/6/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grants R01 AI/RR 37750 (to J. G. F.) and R01CA67463 (to T. C. W. and J. G. F.).

2 To whom requests for reprints should be addressed, at Division of ComparativeMedicine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building 16,Room 825C, Cambridge, MA 02139. Phone: (617) 253-1757; Fax: (617) 258-5708;E-mail: jgfox@mit.edu.

3 The abbreviations used are: MNNG,N-methyl-N9-nitro-N-nitrosoguanidine; CFU,colony-forming unit(s); WPI, weeks post infection; BrdUrd, 5-bromo-29deoxyuridine;TBS, Tris-buffered saline.

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MATERIALS AND METHODS

Animals. One hundred eight, 8-week-old female C57BL mice that werefree of Helicobacterspp,Citrobacter rodentium,Salmonellaspp. endopara-sites, and antibodies to viral pathogens were obtained from Taconic Farms(Germantown, NY). The mice were housed in microisolator caging within anAAALAC-accredited facility and animal use was approved by the MIT AnimalCare and Use Committee.

Bacteria. H. pylori Sydney strain was used for oral inoculation as describedpreviously (29). The organism was grown for 48 h at 37°C under microaerobicconditions on 5% lysed horse blood agar. The bacteria were harvested after48 h of growth; resuspended in PBS; assessed by Gram stain and phasemicroscopy for purity, morphology, and motility; and tested for urease, cata-lase, and oxidase activity.

Experimental Infection. Seventy-two C57BL p.o. infected with 108 CFUH. pylori Sydney strain in 0.3 ml of PBS given three times every other day.Thirty-six control mice were dosed with PBS only. One-half of the infected(n 5 36) and one-half of the control mice (n5 18) were fed a high-salt diet(7.5% versus0.75% Purina Labs Special Formulation, Richmond, IN) for 2weeks prior to the dosing withH. pylori and throughout the experiment. At 4,8, and 16 weeks post challenge, 12 infected and 6 uninfected mice from eachdiet group were euthanized with CO2. Gastric tissues were collected from thecorpus and antrum and used for quantitative urease activity, quantitativeH.pylori culture, and histopathological evaluation.

Quantitative Urease Activity Assay. Gastric samples (1–2 mm2) wereexcised from the midportion of the corpus and the antrum. A quantitativeurease assay was performed as described elsewhere. In brief, the tissues wereincubated in 1 ml of urea broth for 4 h and centrifuged, and duplicate aliquots(200ml) of urea broth from each gastric tissue were placed in microtiter plates.The extent of color change was recorded in an automated ELISA reader at 550nm (30).

Quantitative Culture. The mass of the tissue was determined by subtract-ing the mass of the tube containing media from the mass after tissue was added.The tissue was homogenized with glass tissue grinders, and the homogenatewas diluted 100- and 1000-fold inBrucellabroth containing FCS. One hundredml of each dilution were spread on selective medium: Blood Agar Base no. 2(DIFCO Laboratories, Detroit, MI) supplemented with 5% horse blood (Re-mel, Lenexa, KS), 50mg/ml amphotericin B, 100mg/ml vancomycin, 3.3mg/ml polymyxin B, 200mg/ml bacitracin, and 10.7mg/ml nalidixic acid(Sigma Chemical Company, St. Louis, MO). Plates were incubated microaero-bically at 37°C for 3–5 days. After verification by Gram stain and urease,catalase, and oxidase reactions, theH. pylori colonies were counted and theCFU per gram of tissue calculated. Comparisons between groups were basedon the log concentrations of bacteria.

Histological Evaluation. The tissue examined consisted of a section ofstomach taken from the greater curvature beginning at the squamocolumnarjunction and ending at the gastroduodenal junction. Stomach tissues were fixedin neutral buffered 10% formalin, processed by standard methods, embeddedin paraffin, sectioned at 5mm, and stained with H&E and Warthin-Starry. Theglandular mucosae of the corpus and antrum were examined histologically forinflammatory and epithelial changes and for the presence ofH. pylori. Inflam-mation was distinguished histologically into chronic (lymphohistiocytic) andactive (granulocytic) components. The contributions of both were graded on anascending scale ranging from 0 to 4, based on the intensity, distribution, andconfluence of inflammatory infiltrates. Group data were compared using theMann-Whitney analysis of nonparametric data. At 16 WPI, the extent ofmultifocal glandular atrophy in the proximal mucosa was measured in thehigh-salt diet group and compared with the corresponding glandular region inthe normal diet group. The length of the glandular zone, primarily composedof parietal cells, was measured as a proportion of total mucosal thickness in theproximal corpus with a 103 10 ocular reticle grid. Similar measurements werenot useful in theH. pylori-infected groups because the marked glandularatrophy induced byH. pylori and other gastricHelicobacters in C57BL mice(27, 29) obviated the observation of a further contribution due to the high-saltdiet.

BrdUrd Immunocytochemistry. Animals received a single i.p. injectionof BrdUrd (50 mg/kg) from a freshly made stock solution (5 mg/ml) dissolvedin PBS according to our previously published protocol (27). The mice wereeuthanized 1 h later. At necropsy, a longitudinal section of stomach was taken

from the greater curvature extending from the squamocolumnar junction to thegastroduodenal junction. Samples were placed immediately in cassettes, fixedin 10% neutral buffered formalin, and embedded in paraffin wax. Immuno-histochemical detection of BrdUrd incorporation was performed on 5-mmsections and visualized with a modified avidin-biotin monoclonal antibodyimmunohistochemical technique, which eliminated background signals due tobinding of the secondary antibody to mouse immunoglobulins in the tissuesections. The day prior to the BrdUrd immunodetection, the biotinylatedsecondary antibody (rabbit antimouse IgG) was conjugated to the primaryantibody (mouse monoclonal anti-BrdUrd; Dako Corp., Carpinteria, CA) insolution. The primary and secondary antibodies were mixed together in TBS at1:40 and 1:200 dilutions, respectively, and incubated at 4°C overnight withgentle agitation. Prior to use the following day, a 1:20 volume of normal mouseserum was added to the conjugate solution and incubated at 4°C for 2 h withgentle agitation to quench unbound sites on the secondary antibody. Afterdeparaffinization in xylene and graded ethanol series, the tissue sections werehydrated with PBS and treated with 20mg/ml proteinase K at 37°C for 5 min.Endogenous peroxidase activity in the tissue section was blocked by immers-ing the slides in 1% hydrogen peroxide in methanol. The slides were thenwashed in tap water. The BrdUrd monoclonal antibody identifies only single-stranded DNA. Denaturation of the tissue DNA was achieved by incubation in1 M HCl at 60°C for 8 min. The slides were washed in tap water and then TBSand incubated with 5% normal rabbit serum to block nonspecific binding of thesecondary antibody. The tissue was then incubated with the conjugate ofmouse monoclonal anti-BrdUrd and biotinylated rabbit antimouse IgG for 4 hat room temperature. After washing in TBS, slides were incubated withperoxidase-conjugated streptavidin (1:400 in TBS) and washed in TBS; thelabeled cells were then visualized by the diaminobenzidine reaction. Sectionswere lightly counterstained with hematoxylin. The nuclei of cells at S-phase ofthe cell cycle during thein vivo BrdUrd incorporation phase were stainedbrown.

Quantitation of BrdUrd Incorporation. Quantitation of epithelial prolif-eration based on BrdUrd incorporation was focused on the corpus mucosaadjacent to the squamocolumnar junction (i.e., limiting ridge) and the antrum.The proximal corpus in C57BL mice has been shown to be a target zone forH.pylori-induced gastric lesions. In contrast,Helicobacter-associated lesions inthe antrum are usually limited, although this site is colonized more denselythan the corpus. Regions within the proximal corpus and antrum, in which thegastric pits and proliferative zones were aligned in the plane of section, wereidentified histologically. Some samples were eliminated from this test becauseof the absence of appropriate tissue representation in the sections evaluated.Positively (i.e., brown nuclei) stained epithelial cells were counted in 5–17(mean, 11.3) and 8–31 (mean, 15.1) contiguous glands in the proximal corpusand antrum, respectively. Assessment of epithelial proliferation was based onthe density of BrdUrd-positive epithelial cells per gastric pit. Groups werecompared using the two-tailed Student’st test for unpaired data;P , 0.05 wasconsidered statistically significant.

Gastrin RIA. Plasma gastrin levels (gastrin amidated at the COOH termi-nus) were determined by RIA using rabbit antiserum L2, which reacts similarlywith G17 and G34 (31).

RESULTS

Quantitative Urease. The mean urease activity (absorbance pergram of tissue) at 4 and 8 WPI was significantly increased (P, 0.05)in the corpus and antrum ofH. pylori-infected mice fed the high-saltdiet compared with theirH. pylori-infected counterparts on the normaldiet (Table 1). The mean concentration of urease remained elevated inthe high-salt group at 16 WPI.

Quantitative H. pylori Culture. The mean density ofH. pylori(CFU/g tissue) at 8 and 16 WPI was increased significantly (P , 0.05)in mice fed the high-salt diet compared with their counterparts on thenormal diet. This effect was evident in samples from the corpus (Fig.1) and antrum (Fig. 2). A gradual decline in the mean density ofH.pylori was present in mice on the normal diet. In contrast, the meandensity was relatively stable in the corpus of mice on the high-saltdiet.

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Histopathological Observations.At 16 WPI, mice fed the high-salt diet had multifocal elongation of the gastric pits and reduction inthe parietal cell zone; this change was observed independently ofH.pylori infection (Fig. 3). The apparent increase in gastric pit lengthwas not associated with an increase in total mucosal thickness, indi-cating the increased length of the gastric pits resulted from diminutionof the glandular zone, specifically the parietal cell component. Chiefcells at the base of the glands were not notably affected. Within thehigh-salt diet group, the length of glandular zones in atrophic fociranged from 34 to 82% (mean, 64%), a significant decrease(P , 0.05) compared with the comparable region in the normal dietgroup, which ranged from 75 to 87% (mean, 82%). No statisticallysignificant increases in inflammation scores were observed in unin-fected mice receiving the high-salt diet, compared with their counter-parts on the low-salt diet. Therefore, the effects associated with thehigh-salt diet on the gastric pits and parietal cell numbers occurredindependent of local inflammation in the uninfected mice.

Mice infected with H. pylori typically developed moderate tomarked atrophic gastritis of the corpus at 16 WPI, characterized bychronic active inflammation composed largely of lymphocytes andgranulocytes. The corpus mucosa was characterized by marked loss ofparietal cells and replacement of the normal oxyntic epithelium byhypertrophy and hyperplasia of the mucous epithelium (Fig. 3). Sta-tistically significant increases in granulocyte infiltration (P , 0.02)and chronic inflammation (P , 0.005) were observed inH. pylori-infected mice of both diet groups compared with their uninfectedcounterparts. AmongH. pylori-infected mice, no significant exacer-bation of inflammation was associated with high salt intake.

BrdUrd Analysis. At 16 WPI, BrdUrd incorporation in the prox-imal corpus and antrum was significantly increased (P , 0.005) inuninfected mice fed the high-salt diet compared with those on thenormal diet (Table 2 and Fig. 4). AmongH. pylori-infected mice, asignificant increase (P, 0.05) in antral BrdUrd labeling was also

associated with the high-salt diet. In the proximal corpus, BrdUrdincorporation was also increased significantly (P, 0.05) in associa-tion with H. pylori infection in the normal diet group.H. pyloriinfection in the high-salt diet group was associated with the twohighest BrdUrd labeling densities of the proximal corpus as well as amean increase in BrdUrd incorporation, compared with uninfectedmice on the same diet. However, because of the higher varianceassociated withH. pylori infection, no statistical significance wasassociated with high salt among infected mice. Likewise,H. pyloriinfection with high salt did not induce a significant increase abovehigh-salt diet alone. As suggested by the minimal increase ofH.pylori-associated antral lesions in the C57BL mice, no significantincrease in labeling was observed in the antrum ofH. pylori-infectedmice in either diet group, compared with their uninfected counter-parts.

Gastrin Analysis. Serum gastrin concentrations were significantlyincreased (P, 0.05) in association withH. pylori infection amongmice on the normal diet (Table 3). AlthoughH. pylori infection in thehigh-salt diet group was associated with a mean increase in serumgastrin compared with uninfected mice on the same diet, statisticalsignificance was not achieved. Exposure to the high-salt diet amonguninfected mice resulted in a mean increase compared with uninfectedmice on the normal diet that did not achieve significance. No sub-stantial differences were apparent between the diet groups amongH.pylori-infected mice. Serum gastrin concentrations had no correlationwith epithelial proliferation as assessed by BrdUrd incorporation.

DISCUSSION

Dietary salt has been linked epidemiologically to gastric cancerrisk. Morbidity and mortality rates of gastric cancer in Asia remain thehighest in the world (32). A dietary component that stands out as animportant factor that contributes to Japanese stomach cancer is theintake of highly salted food (21). The incidence of gastric cancer isclearly higher in countries where diets rich in salt [dried fish preservedin salt (3–20%) and soy sauce (19% salt), and pickled foods (13–25%salt)] are consumed in large amounts (12, 33). Daily consumption ofa hot rice gruel seasoned with soybean sauce (salt content, 18%) hasbeen related to high gastric cancer incidence in Japan (33). One studyreported that humans in areas where salted fish was a dietary stapleexcreted as much as 50 g of salt daily in the urine (12). Geographiccomparisons within Japan indicated that the incidence of gastriccancer paralleled the amount of salted foods consumed (12). Theseearlier epidemiological data are supported by recent results from boththe Intersalt Study and a Japanese study (3, 4). Indeed, the Japanesestudy suggested that the relationship betweenH. pylori infection and

Fig. 1. CFU ofH. pylori per gram of tissue-corpus in C57BL/6 mice,u, normal diet;f, high-salt diet.

Fig. 2. CFU ofH. pylori per gram of tissue-antrum in C57BL/6 mice.u, normal diet;f, high-salt diet.

Table 1 Effect of high-salt diet on gastric urease activitya in H. pylori-infectedC57BL/6 mice

Corpus Antrum

Normal diet High-salt diet Normal diet High-salt diet

4 WPI 7.7 (4.9) 35.6 (31.3)b 14.2 (12.8) 36.1 (18.7)c

n 5 12 n 5 12 n 5 12 n 5 128 WPI 9.7 (9.5) 29.9 (25.4)b 11.7 (11.0) 43.6 (32.6)c

n 5 12 n 5 12 n 5 12 n 5 1216 WPI 2.0 (3.4) 14.7 (24.9) 0.8 (1.2) 9.0 (17.0)

n 5 12 n 5 10 n 5 12 n 5 10a Units 5 OD540/gram tissue (SD).b Significant effect associated with high salt,P , 0.05.c Significant effect associated with high salt,P , 0.005.

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gastric cancer may be confounded by salt consumption (34). Thisobservation, if substantiated, is particularly important in Japan, where70% of men 25–34 years of age and 90% of men 55–64 years of agehaveH. pylori infection (35). In addition, DeKosteret al. (36) haveshown that although thein vivo gastric mucosal cell proliferation ratedoes indeed correlate with salt intake, it only does so inH. pylori-positive patients. In contrast, our present findings in salt-fed miceindicate that dietary salt may increase gastric epithelial proliferationindependent ofH. pylori infection.

The role of salt consumption has also been implicated by experi-mental animal models in which animals fed high-salt diets developedgastric mucosal changes. Most of the salt-associated cocarcinogenesisstudies have been performed in rats and mice, in which the concen-trations of sodium salt varied from 0.7 to 20% in drinking water ordiet. For example, mice fed a diet of dried cod containing 7% NaCldeveloped both acute and chronic gastritis (21). Another group ofinvestigators fed excessively salted rice to mice over a period of timeand induced significant glandular atrophy (22). It has been demon-strated that a sodium-deficient diet restricted tumor growth and thatthe antineoplastic activity of certain anticancer agents decreased inmice when given in salted vehicle (37, 38). In addition, in rodentmodels, a concentrated salt diet caused excessive cell replication inthe gastric mucosa, an effect that possibly increases the incidence of

endogenous mutations and potentiates the actions of other carcinogens(13, 39). High salt intake in rodents has been shown to increase theabsorption of known gastric carcinogens, such as polycyclic aromatichydrocarbons (40). Several independent studies have shown that NaClsolution increased the rate of MNNG-induced gastric adenocarcino-mas in rats (13, 15, 41).

The availability of theH. pylori mouse model offers an idealopportunity to directly examine the interaction between dietary saltand H. pylori infection in accelerating gastric injury and promotingthe development of premalignant gastric lesions. The histopatholog-ical observations of this study at 16 WPI are consistent with andextend previous observations of the effect of high salt intake on thegastric mucosa (16–19, 22). Persistent infection withH. pylori Syd-ney strain was achieved in both the high- and low-dose salt diet-treated C57BL mice for the 16-week duration of the study. In themouse model, high dietary salt statistically increased the level ofH.pylori colonization in the body mucosa at all time points evaluated inthe study (i.e., 4, 8, and 16 WPI) based on either quantitative ureaseassay or culture. The increase inH. pylori gastric colonization asso-ciated with the high-salt diet implicates a unique and potentiallysynergistic mechanism. High salt intake may potentiate carcinogene-sis by facilitating colonization and thereby increase the impact ofchronicH. pylori gastritis.

The increased colonization density ofH. pylori could be potentiatedthrough several mechanisms. Interestingly, a preliminary report hasindicated that gastrin appears to be aH. pylori-specific growth factorin vitro. Human gastrin stimulated the growth of eight differentH.pylori isolates in a specific, dose-dependent manner (42). In thepresent study, terminal serum gastrin concentrations were statisticallyincreased inH. pylori-infected mice and elevated in mice fed high-saltdiets. At present, the serum gastrin concentrations do not whollyparallel the colonization data; however, single sample values fromserum offer only an indirect assessment of tissue concentrations. Theresults may also indicate that the observed salt-associated effects

Fig. 3. Gastritis and mucosal changes in theproximal corpus of C57BL/6 mice at 16 WPI fol-lowing high-salt diet andH. pylori infection. A,uninfected mouse on the normal diet. The glandularzone comprises over 75% of the thickness of themucosa.B, uninfected mouse on the high-salt diet.Multifocal elongation of gastric pits in tandem withprominent reduction in the thickness of the glandu-lar zone occurred in response to the high-salt diet.In some atrophic foci, the glandular zone comprises,50% of the thickness of the proximal corpus.Focal inflammation is present in the underlyingsubmucosa.C, H. pylori-infected mouse on thenormal diet. The characteristic gastric pathology ofH. pylori-infected C57BL/6 mice is illustrated. Theglandular epithelium has been replaced by a hyper-plastic, hypertrophic mucous epithelium (arrow).Inflammatory cells are present in the deep mucosaand in the periglandular tissue. The depicted mousehad aH. pylori colonization density of 0.863 106

CFU/g of gastric corpus.D, H. pylori-infectedmouse on the high-salt diet. The glandular epithe-lium is effaced extensively by a hyperplastic mu-cous epithelium. The mucosa is infiltrated diffuselyby inflammatory cells. The depicted mouse had aH. pylori colonization density of 3.813 106 CFU/gof gastric corpus. H&E staining.Bar, 200mm.

Table 2 Effect of high salt and H. pylori infection on gastric pit BrdUrdincorporationa in the proximal corpus and antrum

Proximal corpus Antrum

Normal diet High-salt diet Normal diet High-salt diet

Sham infected 2.7 (1.2) 5.3 (0.8)b 2.7 (1.3) 6.4 (1.9)b

n 5 6 n 5 6 n 5 6 n 5 5H. pylori infected 6.0 (2.7)c 7.4 (5.9) 3.8 (1.2) 6.0 (2.2)b

n 5 7 n 5 8 n 5 9 n 5 7a Mean number of BrdUrd-positive cells per gastric pit (SD).b Significant effect associated with high salt.c Significant effect associated withH. pylori infection.

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occur through an event downstream from gastrin stimulation orthrough a gastrin-independent process.

Another factor in the increase ofH. pylori colonization is theinduction of foveolar hyperplasia in mice fed high-salt diets. Thegastric foveolae, or pits, represent the primary niche and site ofattachment forH. pylori organisms (43). High-salt diets may syner-gize with gastricHelicobacterinfections through expansion of cellswhereH. pylori colonizes. Hyperplasia of the gastric pit epithelium ofthe proximal corpus and the antrum was increased in association withhigh salt intake. The increase in gastric pit length in mice fed thehigh-salt diet corresponded to a statistically significant increase in thegastric proliferation index. The lengthening of the gastric pits was notassociated with an overall increase in mucosa thickness but instead

occurred in tandem with loss of the parietal cells, which line theunderlying glands. The mechanism for these changes is unclear, but itdoes not appear to involve effects on the inflammatory response,which histologically appeared to be unaffected by high-salt dietsalone. Taken together, these observations suggest that the high-saltdiet altered the overall pattern of differentiation of epithelial cellswithin the oxyntic mucosa. In addition to the possibility that gastrindirectly promotesH. pylori colonization, the histological changes mayalso be due, in part, to increases in plasma gastrin levels. In additionto gastrin, other paracrine hormones may be implicated in the pro-gression of mucosal alterations. Previous studies have indicated thattransforming growth factora can influence the decision of gastricstem cells to proceed through a pit rather than a parietal cell pathway.Overexpression of transforming growth factora can lead to a some-what similar picture of foveolar hyperplasia and atrophy (44, 45), andthis mechanism should be investigated further.

Although lengthened gastric pits and parietal cell loss were alsoobserved in the proximal corpus ofH. pylori-infected mice on thehigh-salt diet, the marked atrophic gastritis induced in C57BL/6 miceby H. pylori infection alone obscured the impact of the high-salt diet.In contrast, the relative absence ofH. pylori-associated inflammatorylesions in the antrum permitted the measurement of a similar high-

Fig. 4. Immunohistochemical detection ofBrdUrd in the proximal corpus of C57BL/6 mice at16 WPI following high-salt diet andH. pylori in-fection.A, uninfected mouse on the normal diet. Alimited number of immunopositive cells are presentin a narrow band at the junction of the glandularzone and the overlying gastric pit (arrow). B, un-infected mouse on the high-salt diet. There is anincrease in the density of immunopositive cells pergland in mice on the high-salt diet. Because of theelongation of the gastric pits and reduction in theglandular zone thickness, the proliferative zone isshifted down.C, H. pylori-infected mouse on thenormal diet. Infection withH. pylori induced anincrease in epithelial proliferation. The effacementof the normal glandular zone by the hyperplasticmucous epithelium results in spread of immuno-positive cells to the base of the glands (arrow). D,H. pylori-infected mouse on the high-salt diet. Al-though no statistical difference was observed be-tween the two infected groups, the highest densityof BrdUrd labeling was observed in infected miceon the high-salt diet. Streptavidin-horseradish per-oxidase, 3,39-diaminobenzidiine with hematoxylincounterstain.Bar, 100mm.

Table 3 Effect of high salt and H. pylori infection on serum gastrina

Normal diet High-salt diet

Sham infected 23.7 (9.7) 30.7 (8.5)n 5 6 n 5 7

H. pylori infected 51.8 (29)b 49.7 (42)n 5 12 n 5 11

a Mean concentration [pM] of serum gastrin (SD).b Significant effect associated withH. pylori infection,P , 0.05.

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salt-associated effect on epithelial proliferation in the antral mucosa ofinfected mice. Because local inflammation may confound or exacer-bate the long-term progression of high-salt-associated lesions, infuture studies it would be useful to examine other mouse strains (e.g.,BALB/c) that develop less severeH. pylori-associated gastritis inparallel with C57BL/6 mice.

In conclusion, our study suggests that high-salt diets contribute togastric atrophy and synergize withHelicobacter infections throughfoveolar hyperplasia and expansion ofH. pylori colonization.Whether the effects on gastric differentiation and increasedH. pyloricolonization lead to accelerated progression to gastric cancer is un-known at present but should be addressed in future studies.

ACKNOWLEDGMENTS

Gastrin RIAs were kindly performed by Andrea Varro and Graham J.Dockray.

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EFFECTS OF HIGH-SALT DIET IN C57BL/6 MICE

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1999;59:4823-4828. Cancer Res   James G. Fox, Charles A. Dangler, Nancy S. Taylor, et al.   Colonization in C57BL/6 Mice

Helicobacter pyloriParietal Cell Loss, and Enhances High-Salt Diet Induces Gastric Epithelial Hyperplasia and

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