helicobacter pylori-associated gastric cancer in ins-gas ...pylori infection. also at 8 and 16 wpi,...

[CANCER RESEARCH 63, 942–950, March 1, 2003]

Helicobacter Pylori-associated Gastric Cancer in INS-GAS Mice Is Gender Specific1

James G. Fox,2 Arlin B. Rogers, Melanie Ihrig, Nancy S. Taylor, Mark T. Whary, Graham Dockray, Andrea Varro,and Timothy C. WangDivision of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139 [J. G. F., A. B. R., M. I., N. S. T., M. T. W.]; Gastroenterology Division,University of Massachusetts Medical Center, Worcester, MA [T. C. W.]; and Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom [G. D., A. V.]

ABSTRACT

Previous studies from our group have shown that hypergastrinemia inmice can synergize with Helicobacter felis infection to induce gastriccarcinoma. In addition, epidemiological evidence and a recent study withC57BL/6 mice have strongly suggested a link between a high-salt dietduring Helicobacter pylori infection and the development of hypergas-trinemia and preneoplastic gastric lesions. To address the possible rela-tionship between the two cofactors (gastrin and salt) and whether H. pylorican also lead to gastric cancer in this model, we undertook a longitudinalstudy involving 86 INS-GAS mice. The mice were fed either a high-salt(7.5%) or basal (0.25%) diet, and half were infected with H. pylori.Necropsies at 5 and 7 months postinfection included histopathologicalexamination, quantitative culturing for bacterial colonization levels, andserology to estimate the magnitude of the Th1 and Th2 systemic inflam-matory responses. Lesions consistent with in situ and intramucosal carci-noma were seen in H. pylori-infected male mice only. There was a highlysignificant main effect for Helicobacter infection status for all fundic andantral lesion parameters (P < 0.0001), as well as significant interactions ofinfection status with diet for all of the fundic parameters (all P < 0.03),except intestinal metaplasia. In subsequent ANOVAs in which the datawere limited to that from infected animals, there was a highly significantmain effect for time, diet, and gender (all P < 0.02) on all of the corpuslesion parameters scored (inflammation, atrophy, hyperplasia, metapla-sia, and dysplasia/neoplasia). In addition, gender interacted significantlywith time (all P < 0.03), and. H. pylori colonization increased quantita-tively over the course of the experiment but were independent of eitherdiet or gender. The Th1-associated serum IgG2a responses to H. pyloriincreased from the time of experimental infection to necropsy at 5 or 7months and were similar among all experimentally infected mice with noinfluence of gender (P > 0.10) or dietary salt (P > 0.27). In contrast, theTh2-associated serum IgG1 response to H. pylori was significantly in-creased in infected male INS-GAS mice on the high-salt diet at 7 monthspostinfection (P < 0.012). These results show that H. pylori can alsoaccelerate the development of gastric cancer in the INS-GAS mousemodel, and the results suggest that salt has less of a procarcinogenic effectin the setting of endogenous hypergastrinemia. The increased Th2-asso-ciated humoral response of the infected male mice on the high-salt dietcorrelated with less severe gastric lesions. In the INS-GAS mouse model,male gastric tissue responded more rapidly and aggressively to H. pyloriinfection, high-salt diet, and the combination when compared with fe-males; a finding that appears consistent with the greater incidence ofgastric carcinoma in men. This study highlights the importance of usingboth genders to investigate the pathogenesis of H. pylori.

INTRODUCTION

Both tumor initiators as well as tumor promoters are thought to playan important role in gastric carcinogenesis. The deleterious effects ofsalt, including its role in gastric cancer, is based on epidemiological

studies, biochemical analyses, and in vivo experimentation (1, 2). Anincreased risk of gastric cancer has been associated with the ingestionof salty preserved food (3), as well as preference for salty foods (4, 5),which incorporate the measurement of salt consumption (6–9). Saltadministered p.o. on a weekly basis or fed in the diet during 12–20weeks of exposure to MNNG3 in drinking water promoted the induc-tion of gastric adenocarcinoma in rats (10–12). Ingestion of salt byrats leads to chronic injury of the surface gastric epithelium followedby gastric epithelium proliferation (13–16). Rats given salt in the dietand MNNG in drinking water developed an 80% incidence of adeno-carcinomas in the antrum but not in the corpus (11), whereas ratsgiven a single dose of MNNG [MNNG in water (5 g/liter), 0.25 ml/10g body weight] by intubation and fed a 10% NaCl diet for 1 year hadsignificantly increased tumor burden in both the forestomach andglandular stomach (17). Mice fed a rice diet containing highly saltedfood developed acute gastric mucosal damage (18). Swiss/ICR micefed salted (10% w/w NaCl) rice diets for 3–12 months developedhypertrophy of the forestomach and atrophy (noted by a loss ofparietal cells) of the glandular stomach (19).

We recently fed a high-salt diet (7.5 versus 0.25%) for 16 weeks tofemale C57BL/6 mice infected with H. pylori (20). Half of theinfected and control mice were fed the high-salt diet for 2 weeksbefore dosing and throughout the 16-week experiment. At 16 WPI,mice in both the normal and the high salt diet groups developedmoderate to marked atrophic gastritis of the corpus in response to H.pylori infection. Also at 8 and 16 WPI, cfu/g tissue of H. pylori weresignificantly higher (P � 0.05) in the corpus and antrum of animals inthe high-salt diet group compared with those on the normal diet. Thegastric pits of the corpus mucosa in mice on the high-salt diet wereelongated and colonized by H. pylori more frequently, compared withmice on the normal diet. Collectively, these studies in rodents supportthe hypothesis that salt can contribute to atrophy and function as acocarcinogen.

To additionally investigate interactions between H. pylori, we de-signed an experiment where INS-GAS mice known to develop gastriccancer at 7 months after H. felis infection (21) were infected with H.pylori and fed a high-salt diet to determine whether infection andelevated dietary salt increased the severity of gastric lesions in thesemice. We also included male and female mice in the study to deter-mine whether gender influenced disease severity.

MATERIALS AND METHODS

Animals. Eighty-six (43 male and 43 female) weanling, specific pathogen-free INS-GAS mice on a FVB background (free of enteric Helicobacter spp,Citrobacter rodentium, Salmonella spp, endoparasites, ectoparasites, and se-rum antibodies to murine viral pathogens) obtained from an in-house breedingcolony were used in the study (Table 1).

Animals were housed in microisolator, solid-bottomed polycarbonate cagesand fed a commercially prepared pelleted diet and given water ad libitum. Themice were all maintained in an Association for Assessment and Accreditationof Laboratory Animal Care-approved facility under barrier conditions as virusantibody-free mice for the duration of the 7-month experiment. The protocol

Received 9/26/02; accepted 1/6/03.The costs of publication of this article were defrayed in part by the payment of page

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

1 Supported by NIH Grants T32-RR07036 and AI37750 (to J. G. F.) and NIH R01CA93405 (to T. C. W.).

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) 252-1877;E-mail: [email protected].

3 The abbreviations used are: MNNG, N-methyl-N�-nitro-N-nitrosoguanidine; WPI,weeks postinfection; cfu, colony-forming unit.

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was approved by the Animal Care Committee of the Massachusetts Institute ofTechnology.

Bacteria. H. pylori Sydney strain was used for oral inoculation as de-scribed previously (22). The organism was grown for 48 h at 37°C undermicroaerobic conditions on 5% lysed horse blood agar. The bacteria wereharvested after 48 h of growth, resuspended in PBS, assessed by Gram’s stainand phase microscopy for purity, morphology, and motility and tested forurease, catalase, and oxidase activity.

Experimental Infection. Forty-eight (divided equally among female andmale) INS-GAS mice were p.o. infected with 108 cfu H. pylori Sydney strainin 0.3 ml of PBS given three times every other day. Thirty-eight control micewere dosed with PBS only. Half of the infected and half of the control micewere fed a high-salt diet (7.5 versus 0.75% Purina Labs, special formulation)for 2 weeks before the dosing with H. pylori and throughout the experiment(20). At 5 and 7 months after challenge, 12 infected and 4–6 uninfected micefrom each diet group were euthanatized with CO2 (Table 1). Gastric tissueswere collected from the corpus and antrum and used for quantitative H. pyloriculture and histopathological evaluation.

Quantitative Culture. The weight of the gastric tissue was determined bysubtracting the weight of the tube containing media from the weight afteradding tissue. The tissue was homogenized using glass tissue grinders and thehomogenate diluted 100- and 1000-fold in Brucella broth containing FCS. Onehundred �l of each dilution was spread on selective medium: blood agar baseno. 2 (Difco) supplemented with 5% horse blood (Remel) and 50 �g/mlamphotericin B, 100 �g/ml vancomycin, 3.3 �g/ml polymyxin B, 200 �g/mlbacitracin, and 10.7 �g/ml naladixic acid (Sigma Chemical Company, St.Louis, MO). Plates were incubated microaerobically at 37°C for 3–5 days.After verification of H. pylori by Gram’s stain, urease, catalase and oxidasereactions. Bacterial colonies were counted and the cfu/g of tissue calculated.Comparisons between groups were based on the log concentrations of bacteria.

Histological Evaluation. At necropsy, the stomach and proximal duode-num were removed and incised along the line of the greater curvature. Luminalcontents were removed and the mucosa rinsed with sterile saline. For histopa-thology, linear strips extending from the squamocolumnar junction throughproximal duodenum were taken along the lesser curvature, placed on a card,and fixed in either 10% neutral-buffered formalin or Carnoy’s fixative. Tissueswere routinely paraffin embedded, cut at 5 �m, and stained with H&E forhistopathology.

Immunohistochemistry. Mice received a single infection of BrdUrd at adose of 5 mg/�g i.p. 1 h before euthanasia (20). Unstained, deparaffinized,Carnoy’s-fixed sections were stained for BrdUrd incorporation as describedelsewhere (20). Additionally, formalin-fixed sections were antigen retrievedwith pepsin (Zymed, San Francisco, CA) for 10 min at 37°C and were labeledwith a polyclonal rabbit antibody recognizing the cleaved (activated) form ofhuman caspase-3 (Cell Signaling Technologies, Beverly, MA) or rat mono-clonal antibody recognizing mouse leukocyte common antigen (CD45, Ly-5;BD PharMingen, San Diego, CA). Primary antibody binding was detected withspecies-appropriate biotinylated secondary antibodies (Sigma Chemical Com-pany), streptavidin peroxidase, and 3,3�-diaminobenzidine (Vector Laborato-ries, Burlingame, CA), and tissues were counterstained with methyl green(Vector Laboratories). Immunohistochemical assays were performed on anautomated immunostainer (i6000; Biogenex, San Ramon, CA).

Quantitative Morphometry for Cell Proliferation. BrdUrd-labeled cellnumbers were compared between groups at the 7-month time point by mor-phometric analysis. Images of tissue sections stained for BrdUrd by immuno-histochemistry (described above) were captured with a DXM1200 digital

camera (Nikon Instruments, Melville, NY) and morphometric analysis wasperformed using IPLab 3.5 software for Macintosh (Scanalytics, Inc., Fairfax,VA). Three images of well-oriented orad gastric corpus/animal were analyzed.The gastric mucosa was manually outlined with an electronic drawing tablet(Graphire 2; Wacom Technology, Vancouver, WA), and the bounded area wasdefined as the region of interest. Color thresholding was used to selectivelyhighlight labeled cells (brown, against a green background).

Serology. Evaluation of serum antibody responses to H. pylori. Serum wascollected at 2, 4, 5, and 7 months and evaluated by ELISA for serum IgG2a(Th1-promoted isotype) and IgG1 (Th2-promoted isotype) using an outermembrane antigen preparation of H. pylori as described previously (24).Antigen was coated on plates at a concentration of 10 �g/ml, and sera werediluted 1:100. Biotinylated secondary antibodies included monoclonal anti-mouse antibodies produced by clones G1-6.5 and R19-157 (BD PharMingen)for detecting IgG1 and IgG2a, respectively. Incubation with extravidin perox-

Fig. 1. Gastric histopathology (cardia and orad corpus) demonstrating more severehyperplasia and dysplasia in H. pylori-infected male mice than females. A similar but lesspronounced gender effect was observed in uninfected mice (data not shown). Basal versushigh-salt diet had no significant effect on lesion scores. Samples from mice harvested at5 months postinoculation. (a) female, basal diet; (b) male, basal diet; (c) female, high-saltdiet; and (d) male, high-salt diet. H&E, original magnification: �100.

Fig. 2. Gastric histopathology demonstrating significantly more severe inflammation,hyperplasia, and dysplasia in mice infected with H. pylori versus uninfected age-matchedcontrols. Samples are from male mice collected at 7 months. a, uninfected, basal diet; b,infected, basal diet; c, uninfected, high-salt diet; and d, infected, high-salt diet. H&E,original magnification: �100.

Table 1 INS-GAS mice infected with H. pylori and fed basal or high-salt diet

Time and diet

5 months 7 months

Basal High salt Basal High salt

H. pylori infected miceMale 6 6 6 6Female 5 6 6 6

Uninfected control miceMale 4 5 4 6Female 4 5 4 6

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HELICOBACTER PYLORI-ASSOCIATED GASTRIC CANCER



idase (Sigma Chemical Company) was followed by ABTS substrate (Kirke-gaard and Perry Laboratories, Gaithersburg, MD) for color development.Absorbance development at 405� was recorded by an ELISA plate reader(Dynatech MR7000; Dynatech Laboratories, Inc., Chantilly, VA).

Statistical Analysis. An ANOVA was performed to examine the effect ofhelicobacter infection, gender, high-salt diet, and duration of infection (time)on the corpus and antral gastric lesions, helicobacter colonization, and sero-logical responses of INS-GAS transgenic mice. In addition to the level ofcolonization in each of the gastric compartments, there were five histopatho-logical measures of disease assessed in the corpus; inflammation, glandularatrophy, hyperplasia, metaplasia and dysplasia/neoplasia, and two antral meas-ures; inflammation and hyperplasia that were assessed. The ANOVA model foreach disease marker contained all independent variables and significant two-way interaction terms.

To investigate the influence of gender, diet and time on the pathogenesis ofhelicobacter-induced disease in more depth, a second ANOVA was performedfor each lesion variable using the data from infected animals only. Graphs ofthe significant interactions were constructed to study the relationship of allinteractions in greater detail. The colonization data were log transformedbefore any analyses. They were then subjected to analysis with the sameANOVA model as that used to analyze the histopathology scores. For BrdUrdanalysis, percentages of the region of interest (i.e., gastric mucosa) containing

BrdUrd label were tabulated and analyzed statistically using Prism 3.0 soft-ware for Macintosh (GraphPad, San Diego, CA). Mean differences betweengroups were assessed by one-way ANOVA, with values � 0.05 consideredsignificant. Comparisons between pairs of groups were performed using theStudent t test.

Gastrin Radioimmunoassay. Plasma gastrin levels (COOH-terminallyamidated gastrin) were determined by radioimmunoassay using rabbit anti-serum L2 that reacts similarly with G17 and G34 (23).

RESULTS

At the 5-month collection point, both infected and uninfectedINS-GAS mice on either the basal or high-salt diet exhibited minimal-to-mild hyperplasia and dysplasia in the females and mild-to-moder-ate changes in the males. However, this gender effect was morepronounced in the infected mice (Fig. 1). Unlike gender, diet exertedno significant effect. Additionally, there was moderate loss of chiefcells and mild loss of parietal cells with equivalent gender-specificseverity among these groups. At the 7-month time point, uninfectedINS-GAS mice demonstrated more severe hyperplasia, dysplasia, andchief and parietal cell atrophy than at the 5-month time point, with anequivalent gender effect (Fig. 2, a and c). Again, there was noappreciable difference between the uninfected mice fed the basal dietversus those given the high-salt diet. H. pylori infection significantlyexacerbated disease in all groups (Fig. 2, b and d). Surprisingly, lesionscores in the mice on the basal diet were slightly higher than those onthe high salt diet among infected mice. Lesions consistent with in situand intramucosal carcinoma were only observed in male mice in thebasal diet group. Four of 6 H. pylori-infected male mice on the basaldiet, infected for 7 months, were diagnosed with gastric cancer, and allremaining infected male mice on both diets exhibited severe dysplasiaand preneoplastic lesions (Fig. 3).

H. pylori infection in mice resulted in gastric mucosal inflamma-tion, epithelial hyperplasia, metaplasia, and dysplasia (Fig. 4).Changes included elongated branched and tortuous glands, cystic

Fig. 3. Comparison of gastric inflammation and dysplasia scores in female and maleINS-GAS mice on a basal or high-salt diet and infected with H. pylori for 7 months.

Fig. 4. Gastric histopathology demonstrating in-flammatory, hyperplastic, metaplastic, and dysplas-tic changes in mice infected with H. pylori. a,ectatic gland/crypt lined by attenuated epitheliumand filled with viable and degenerate neutrophilsand sloughed epithelial cells (crypt abscess). b,numerous apoptotic cells with clear or condensedcytoplasm and shrunken, condensed, frequentlyfragmented nuclei. Many apoptotic cells and cellfragments have been ingested by adjacent epithelialcells. c, epithelial dysplasia with elongation,branching, and tortuosity of glands and multifocalcystic dilation with intraluminal cellular debris. d,intestinal metaplasia characterized by microvillousbrush borders (inapparent at this magnification),columnar elongation, and large round to oval palecytoplasmic vacuoles (goblet metaplasia). e, mu-cous metaplasia of parietal cells. Note foamy cyto-plasm and basal margination and condensation ofnuclei. f, signet ring epithelial cell forms (arrows)in dysplastic epithelium. g, papillary infolding andnuclear piling in hyperplastic and dysplastic epithe-lium. h, mitotic figures, including bizarre form(arrow). i, intramucosal carcinoma in H. pylori-infected male on basal diet. Dysplastic glands haveinvaded the muscularis mucosae, abut submucosallymphatics (bottom), and are separated only by alayer of lymphatic endothelium. H&E, originalmagnification: �200 (a, b, d, and g); �100 (c andi), and �400 (e, f, and h).

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dilation with crypt abscesses, and pseudostratification up to severalcells deep. More severely affected animals exhibited multifocal intes-tinal metaplasia characterized by epithelial columnar elongation, mi-crovillous brush borders, and single large cytoplasmic round vacuoles.Within dysplastic foci were single apoptotic cells characterized bydiffusely granular to hyaline eosinophilic cytoplasm, karryorhexis,and nuclear pyknosis. Phagocytosed apoptotic cells or their remnantswere common in cytoplasmic vacuoles in neighboring epithelial cells.Transition zones from normal to hyperplastic to dysplastic epitheliumwere usually discernible and occurred in a wave of decreasing severityaborally from the cardia such that lesions were invariably most severeadjacent to the cardia. Moreover, there was severe chief cell atrophythat was complete in some males and losses up to 50–75% of parietalcells. In some mice, chief cell areas displayed a mucous neck cellphenotype, suggesting dedifferentiation to a more primordial state orabsence of differentiation during downward migration. Parietal cells,sometimes in large numbers, exhibited small vacuolar to foamy meta-plasia, taking on an appearance reminiscent of duodenal submucosal(Brunner’s) gland cells (Fig. 4).

Dysplastic changes supportive of a diagnosis of carcinoma in situincluded intraglandular folding and papillary and ductular projections,moderate to marked cellular plemorphism, cellular atypia, includingsignet ring forms, euchromatic nuclei with up to two to three mitotic

figures/high power field, and occasional bizarre mitotic figures (Fig.4). Two animals exhibited features of intramucosal carcinoma, withglandular invasion into the muscularis mucosae. Dysplastic glandsabutted submucosal lymphatics (Fig. 4), but frank submucosal inva-sion was not demonstrated.

Immunohistochemistry. Leukocyte common antigen (CD45) la-beling (Fig. 5, a and b) subjectively was directly proportional toinflammation scores assigned to H&E-stained sections. Likewise,BrdUrd labeling was proportional to mucosal epithelial hyperplasiascores (see below), and mitotically active cells were encountered bothabove and below the normal proliferative zone, with disorganizationmirroring the overall level of mucosal dysplasia (Fig. 5, c and d).Cleaved (activated) caspase-3 was detected in surface (terminal) ep-ithelial cells regardless of degree of dysplasia or cellular atypia (Fig.5, e and f), suggesting that dysplastic cells in this model retained thefunctional cell signaling pathways necessary for terminal apoptosis.Marked cellular pleomorphism and variation in surface epithelialthickness precluded meaningful quantitative comparison of the num-ber of caspase-3-positive cells in dysplastic stomach sections; how-ever, the relatively uniform immunolabeling of surface epitheliumcorroborated the widespread finding of apoptotic cells in H&E-stainedsections.

Fig. 5. Gastric immunohistochemistry. FewerCD45� inflammatory cells in uninfected (a) versus H.pylori-infected (b) mouse. Fewer BrdUrd� dividingcells in stomach from uninfected (c) mouse versusmouse with carcinoma in situ (d). Demonstration ofcells undergoing apoptosis by labeling with antibodyspecific for cleaved (activated) caspase-3. Cells near thesurface of uninfected mouse contain activatedcaspase-3, as do epithelial cells from mouse with severedysplasia, suggesting that dysplastic cells retain func-tional apoptotic pathways. Immunoperoxidase tech-nique, 3,3�-diaminobenzidine chromogen, methyl greencounterstain; original magnification: �100.

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Quantitative Morphometry for Cell Proliferation. Mice infectedwith H. pylori had more BrdUrd-labeled cells (i.e., proliferation)/unitarea of gastric mucosa than did uninfected mice (Fig. 5). This differ-ence in BrdUrd labeling between groups was highly significant sta-tistically (P � 0.0001). The difference was significant in both sexes,although males (P � 0.0001) demonstrated larger differences betweeninfected and uninfected cohorts than did females (P � 0.035). Strat-ified by gender and diet, there was a statistically significant difference(P � 0.05) in every group between uninfected and infected mice,except females on the basal diet (P � 0.14), probably because of thelarge SD in the uninfected females on the basal diet. However, therewere no statistically significant differences in BrdUrd labeling be-tween males versus females or basal versus high-salt diet groupsbetween mice with the same infection or noninfection status. Thus, theonly risk factor associated with statistically significantly increasednumbers of BrdUrd� nuclei (i.e., cell proliferation) in the gastricmucosa was H. pylori infection.

Serum Antibody Responses to H. Pylori. The Th1-associatedserum IgG2a response to H. pylori increased from the time of exper-imental infection to necropsy at 5 or 7 months (Fig. 6a). At 2, 4, 5, and7 months postinfection, the IgG2a responses were similar among allexperimentally infected mice with no influence of gender (P � 0.10)or salt content of the diet (P � 0.27). In contrast, the Th2-associatedserum IgG1 response to H. pylori was significant by 2 monthspostinfection (P � 0.05) but plateaued in most groups of experimen-tally infected mice through the time of necropsy (Fig. 6b). The IgG1response at 7 months postinfection in infected male INS-GAS mice onthe high-salt diet was significantly higher than the infected males onthe low-salt diet or infected females on either salt diet (P � 0.012).

Statistical Analysis. A series of ANOVAs was performed to de-termine the significance of H. pylori infection in the development ofgastric lesions. There was a highly significant main effect for helico-bacter infection status for all fundic and antral lesion parameters(P � 0.0001), as well as significant interactions between infectionstatus and diet for all of the fundic parameters (all P � 0.03) with the

exception of intestinal metaplasia. In contrast, although serum gastrinwas highly correlated with fundic inflammation, hyperplasia, glandu-lar atrophy, metaplasia, and dysplasia (all P � 0.004), there was noeffect on gastrin levels associated with infection status (P � 0.4).There was a main effect for diet and time (all P � 0.01), although notfor gender (P � 0.4), on serum gastrin. This was a reflection of thesignificantly higher gastrin levels observed at the later time point andthe fact that gastrin levels were generally lower in animals on the highsalt diet (Fig. 7).

In subsequent ANOVAs in which the data were limited to that frominfected animals, there was a highly significant main effect for time,diet, and gender (all P � 0.02) on all of the corpus lesion parametersscored (inflammation, atrophy, hyperplasia, metaplasia, and dyspla-sia/neoplasia). Gastric pathology was more severe in males, increasedwith time and, surprisingly, was milder in animals on the high-saltdiet. In addition, gender interacted significantly with time (allP � 0.03), which prompted additional exploration into the nature ofthese interactions.

The significant two-way interactions are depicted graphically inFigs. 8 and 9. When infection status is included as a variable, there isa significant diet by infection interaction (Fig. 8). Interestingly, for allfundic parameters, the high-salt diet results in an increase in pathologyin the uninfected animals and a marked decrease in the infectedanimals. When only infected animals are considered in the analyses,there is a significant time by gender interaction (Fig. 9). Althoughtime is an important factor in the pathogenesis of disease for bothmales and females, females have a delayed response to helicobacterinfection relative to the males, reflected in the lower lesion scores atthe 5-month time point but then show a greater increase in the severityof disease over time, as indicated by the steeper slopes of the femalecurves.

H. Pylori Colonization. The single factor that significantly influ-enced colonization in the corpus and the antrum was duration ofinfection (all P � 0.0001; Fig. 10). Colonization of both the corpusand antrum, which occurred in all infect mice, was comparable at the5-month time point and colonization in both of these gastric compart-ments increased significantly within the 2-month time frame of thestudy regardless of gender or diet.

Fig. 6. Serum was tested by ELISA at 2–7 months PI for Th1 and Th2-associatedantibody responses to H. pylori antigens. The Th1 response (A) was similar among allgroups of mice, but the Th2 response (B) of the infected males fed high salt wassignificantly higher at 7 months postinfection.

Fig. 7. Serum gastrin levels observed in mice on the basal versus the high-salt diet ateach time point in infected and uninfected mice. Gastrin increased significantly with timeand was generally lower in animals on the high-salt diet. This difference was mostdramatic in the infected animals at the 7-month time point. The error bars in the graphsrepresent 95% confidence intervals for the group means.

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DISCUSSION

In this study, we used the hypergastrinemic INS-GAS mouse modelto study the effect of H. pylori infection and a high-salt diet on gastriccancer progression. We were able to document H. pylori associatedgastric cancer in INS-GAS mice. This appears to be the first report ofthe induction of gastric cancer by H. pylori in any mouse model andthus confirms the carcinogenicity of this organism. Moreover, thedevelopment of gastric cancer over the 7-month observation periodwas seen only in INS-GAS H. pylori-infected male mice on thelow-salt diet.

We previously demonstrated that elevations in gastrin-17 levelshave also been associated with an increased risk for gastric glandularatrophy and cancer (21). Elevation in levels of G-17 in INS-GAStransgenic mice on a FVB background, which we also used in thisstudy, resulted in the spontaneous development of atrophy and gastriccancer in uninfected mice over a period of 1–2 years and more rapiddevelopment of cancer (7 months) in H. felis-infected mice (21). Ourcurrent study demonstrates that H. pylori can likewise induce gastriccancer in male but not female INS-GAS mice within 7 monthspostinoculation that is independent of dietary salt intake. Dysplasticand neoplastic changes observed in this study, which mirror lesionsdescribed in H. felis-infected INS-GAS mice and which are features ofH. pylori-associated adenocarcinoma in humans and gerbils, includeepithelial dysplasia, papillary and ductular hyperplasia, cellular aty-pia, and bizarre mitotic figures (25–27). Signet ring forms seen in theH. pylori-infected mice with gastric tumors is also seen in H. pylori-associated gastric adenocarcinoma of humans but was not noted in H.

felis INS-GAS gastric tumors (21). However, tumors in H. pylori-infected INS-GAS mice covered less gastric surface area than didtumors induced by H. felis infection (unpublished observations), andvascular invasion was documented in H. felis- but not H. pylori-infected INS-GAS mice by 7 months postinfection (21). This variationin disease severity may reflect a more robust adaptation of H. felis tothe murine stomach or may be the result of inherent differences inbacterial pathogenicity between the two strains [although H. felis isCagA (�) and VacA (�)].

Our study also supports a role for H. pylori-mediated hyperprolif-eration in the induction of gastric cancer. Immunohistochemical mark-ers of cellular proliferation (BrdUrd incorporation) and apoptosis(activated caspase-3) demonstrated significantly increased mitotic ac-tivity associated with H. pylori infection. Although by morphometricanalysis, males demonstrated higher BrdUrd labeling indices than didfemales, these differences did not reach statistical significance, prob-ably because of individual biological and histological sampling vari-ation. Subjective interpretation of caspase-3 labeling revealed nodifferences in the ability of epithelial cells to undergo apoptosis,regardless of hyperplasia or dysplasia status. Taken together, thesefindings suggest that the gastric epithelial layer thickening associatedwith H. pylori infection is attributable more to increased cell prolif-eration than to decreased apoptosis. Peek et al. (28) have shown thatH. pylori infection in gerbils leads to increases in gastric epithelialproliferation that appear to be temporally related to increased serumgastrin levels. This group concluded that the epithelial cell growth inH. pylori-colonized mucosa of the Mongolian gerbil may be mediated

Fig. 8. Set of graphs depicting significant “Infection by Diet” interactions that were observed for all corpus lesion parameters with the exception of intestinal metaplasia.

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by gastrin-dependent mechanisms. Thus, the two animal models de-scribed to date that appear most susceptible to Helicobacter-depend-ent gastric cancer—the INS-GAS mouse and the Mongolian gerbil—are both characterized by markedly elevated levels of amidatedgastrin. In addition, at least one study has shown that H. pylori-infected patients elevated levels of amidated gastrin at baseline aremore likely to progress to glandular atrophy after treatment withproton pump inhibitors (29).

In our study, gastric cancer was observed only in the infected maleINS-GAS mice. Previous studies have shown that susceptibility toHelicobacter-mediated atrophy and preneoplasia is strongly influ-enced by genetics, but little attention has been paid to gender althoughmale humans are at increased risk to develop gastric cancer. Forexample, several groups have demonstrated that C57BL mice have avigorous Th1-mediated gastric inflammation after infection with He-licobacter spp, whereas the BALB/c mouse has minimal gastricinflammation (indicative of a Th2 response). In general, there is aninverse relationship between the strength of the Th1 response and thelevel of Helicobacter colonization (30). The cell-mediated immunehyporesponsiveness to gastric helicobacter infection observed in dis-ease-resistant strains such as BALB/c and CBA mice appears to be adominant trait, as evidenced by results from analysis of the gastricphenotype in F1 generation of CBA/c x C57BL infected with gastrichelicobacters (31, 32). These investigators had also used both femalesand males but compared with our 7 month study, their study periodwas for 3 months, and a gender effect was not noted (31, 32). Indeed,many studies use female mice based on the practicality that cohousedfemale mice are less likely to fight when compared with cohousedmale mice.

Recently, however, a gender effect in mice was described in an oralvaccination trial using interleukin receptor 4� chain-deficientBALB/c mice in which H. pylori native urease (5 �g) and choleratoxin (10 �g) administered at weekly intervals for 4 weeks providedprotection against 109 cfu of mouse-adapted P76 H. pylori (33) Theseauthors noted higher colonization rates in H. pylori-infected controlmale interleukin receptor 4� mice and wild-type male BALB/c micethan in their female counterparts (33). This finding was statisticallysignificant in interleukin receptor 4��/� mice. Unfortunately, his-topathological correlates of gastric disease between male and femalemice were not presented (33). Our study documents clearly the greatersusceptibility of male gastric tissue to H. pylori infection, a findingconsistent with the higher rates of gastric carcinoma in men. TheINS-GAS mouse model will likely be useful in additional investiga-tions that will address the mechanisms responsible for this genderdifference in susceptibility.

The most surprising finding was perhaps the absence of synergismin the INS-GAS mouse model between H. pylori infection and ahigh-salt diet. In an earlier study performed in H. pylori-infectedC57BL/6 mice, we demonstrated that a high-salt diet accelerated thedevelopment of hyperproliferation, pit cell hyperplasia, and glandularatrophy (20). However, this C57/BL mouse study was terminated at16 WPI, rather than the 7-month observation period in the currentstudy (20). Nevertheless, we speculated that chronic salt intake mayexacerbate gastritis by increasing H. pylori colonization density andtheorized that the increased colonization might be attributable toseveral mechanisms (20). Terminal serum gastrin concentrations werestatistically increased in H. pylori-infected C57BL mice on a normaldiet versus uninfected controls (51.8 versus 23.7 pm) and elevated in

Fig. 9. Set of graphs illustrating the pattern of significant “Gender by Time” interactions seen in the fundic lesions of infected animals.

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infected mice fed high-salt diets when compared with control mice onthe same diet (20), although the serum gastrin concentrations did notwholly parallel the colonization data. Previous reports had suggestedthat high-salt diets can be associated with hypergastrinemia. Takentogether, our results raised the possibilities that the observed salt-associated effects occurred in part through the induction of gastrin(20).

In this study, we noted an increase in H. pylori colonization densityin INS-GAS mice over time that was independent of salt intake orgender, and we found that the high-salt diet showed a clear-cutprotective effect in this animal model. Only the infected INS-GASmale mice on a low-salt diet developed gastric cancer. Overall, theINS-GAS/H. pylori mice on a high-salt diet showed decreased histo-logical progression to atrophy and neoplasia, which was also reflectedby lesser increases in serum gastrin levels, suggesting better preser-vation of acid secretion and parietal cell function. Possible explana-tions for these different findings include differences in murine geneticbackground in the two studies and the unique characteristics of theINS-GAS mice. Although the possibility cannot be excluded thatFVB/N mice respond differently than C57BL/6 mice to a high-saltdiet, a more plausible explanation relates to the robust nature of theINS-GAS/H. pylori model. It is likely that the unique and rapidprogression to atrophy, metaplasia, and cancer seen in the H. pylori-infected INS-GAS mouse has eclipsed the procarcinogenic effect ofthe high-salt diet, thereby unmasking protective effects that wouldhave otherwise been obscured. The absence of a additive effect byhigh salt in this hypergastrinemic model system strongly suggests that

high salt in wild-type mice, e.g., C57BL mice, is acting, in part,through induction of hypergastrinemia (20). In INS-GAS mice, how-ever, there is already a sufficient elevation of circulating gastrin suchthat high salt no longer has a potentiating effect. In addition, it islikely that high salt is less able to induce gastrin release in these micebecause antral gastrin expression is highly suppressed by circulatingpancreatic gastrin in the INS-GAS transgenic model. Thus, the ab-sence of a potentiating effect by high salt in the INS-GAS model ismost consistent with a model in which gastrin is a downstream targetof high salt.

The mechanism by which high salt exerts its protective effect in theINS-GAS/H. pylori model is currently unknown. However, it is ofinterest that the high-salt diet in infected male mice was associatedwith an enhanced anti-inflammatory Th2 humoral immune responseto H. pylori antigens, which correlated with less severe gastric lesionsand the absence of clear neoplastic transformation at 7 months postin-fection. Mouse strains exhibiting weak type-1 responses to H. pyloriexhibited a significant Th2 cytokine response to helicobacter antigens,and these findings support our observation of an enhanced Th2humoral immune response in the infected males fed a high-salt diet for7 months postinfection. The possibility that high salt is able tomodulate the immune and gastric cytokine response (e.g., interleukin1�) to H. pylori no doubt bears additional investigation.

In summary, we did not see, as expected, a synergistic effect withsalt and H. pylori in the INS-GAS mouse. This supports the notionthat in the mouse model, constitutive hypergastrinemia and highdietary salt intake are nonadditive risk factors for development of H.

Fig. 10. Set of graphs displaying the “Log of the Number of cfu/g of Tissue” in the corpus and antrum of males and females. The only factor that had a significant effect on thelevel of colonization in the corpus and the antrum was time.

949




pylori-induced adenocarcinoma. This result is supportive of the modelthat gastrin acts downstream of and mediates the effects of high saltin the acceleration of gastric cancer, but additional studies are neededto clarify this issue. However, to our knowledge, this is the first studydocumenting a statistical difference in severity of gastritis with pro-gression to gastric cancer in male versus female mice infected with H.pylori. Additional work is needed to determine whether the male-associated risk factors leading to gastric tumors in INS-GAS miceinfected with H. pylori are the same as those in male A/J mice infectedwith H. hepaticus, which are more susceptible to hepatitis and hepa-tocellular carcinoma than their female counterparts (34). Our findingsin the H. pylori-infected male INS-GAS mice is consistent with agreater incidence of gastric cancer in men and highlights the potentialof using H. pylori-infected INS-GAS mice to explore gender-depen-dent mechanisms of gastric carcinogenesis.

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2003;63:942-950. Cancer Res James G. Fox, Arlin B. Rogers, Melanie Ihrig, et al. Mice Is Gender Specific

-associated Gastric Cancer in INS-GASHelicobacter Pylori

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