Summary. We developed a chronic drinking rat modelto investigate the long-term effects of ethanol feeding oncell proliferation and apoptosis in rat stomach. Adultmale Sprague-Dawley (SD) rats received either anisocaloric control or drinking water containing 6% (v/v)ethanol as their only water intake for 1, 3, 7, 14 and 28days. At the end of each feeding period, animals weresacrificed and the stomach was dissected for the samplepreparation. The cell proliferation and apoptosis ingastric mucosa of rats in different groups were analyzedby flow cytometer, immunohistochemistry and computerimage analysis. In the flow cytometric study, comparedwith the control, the cell apoptosis in gastric mucosa ofthe rats was enhanced during the exposure to the ethanolin 3rd to 28th day. Otherwise the cell proliferation wasincreased in 3rd to 14th days, and decreased in 28thdays, respectively. The results were confirmed byimmunohistochemistry and computer image analysisstudied. This finding suggested that short-term chronicadequate alcohol intake may enhance the cell turnover ofgastric mucosa. Long-term stimulus with the lowconcentration ethanol may cause the impairment of thecell turnover function of the gastric mucosa and may beone of the mechanisms underlying the gastric pathologyassociated with alcohol abuse. Key words: Apoptosis, Proliferation, Ethanol, Stomach,Rat

Introduction

Ethanol is one of the most widely used and abuseddrugs. Excess alcohol consumption may induce oraggravate various types of disease. In particular, it isregarded as an important factor for ulcerative andinflammatory lesion gastric mucosa (Luis, 2000).However, the pathogenesis of this lesion remains poorlyunderstood. The gastric epithelial cells are continuously

renewed in the whole extension of the gland (Alvares,1992; Alvares and Gama, 1993).The homeostaticregulation of cell numbers in normal tissues reflects ahighly regulated balance between cell proliferation andcell death (McDonnell, 1993).The imbalance of cellproliferation and apoptosis has been implicated in thepathogenesis of gastric mucosal lesions (Thompson,1995; Potten and Booth, 1997). Chronic ethanolconsumption resulting in gastric mucosal lesions mightthus be expected to influence the kinetic balancebetween cell proliferation and cell death. Some reportshave indicated that ethanol-associated gastric cellproliferation may involve changes in epidermal growthfactor receptors (Wang et al., 1996, 1997) and oxidativestress and lipid peroxidation (Hernandez-Munoz et al.,2000).

Animal investigations with various kinds of ethanolfeeding have been performed. For example, Lieber andDeCarli (1994) reported that ethanol was fed in the dietwith 5% concentration (w/v). Turchan et al. (1999)added the ethanol in the drinking water with theconcentration of 6% (v/v). In this study, we havedeveloped a chronic drinking rat model with continuousethanol ingestion for 28 days using a modified methodof Turchan’s (Turchan et al., 1999). Using this model,we investigated the relationship between chronic ethanolintake and the cell turnover related to cell proliferationand apoptosis. The cell proliferation and apoptosis wereanalyzed by flow cytometer. To confirm the cytometricresults, we also investigated the expression of cellproliferation and the apoptosis marker in the rat gastricmucosa. For this purpose, double-staining using theantibody to proliferating cell nuclear antigen (PCNA)and cytokeration 18 were performed in the same slideand computer image analysis was used to quantity thePCNA and cytokeration 18 labeling. Materials and methods

Animals and treatments

Male Sprague-Dawley rats (200-250g) were used inthis study. Sixty rats were housed in plastic cages in an

Chronic ethanol feeding alters the epithelial cell proliferation and apoptosis in rat gastric mucosaY.B. Ge, J. Du, L.L. Fan. Y.C. Li and L. GuDepartment of Physiology, Nanjing Medical University, Nanjing, China

Histol Histopathol (2007) 22: 185-190

Offprint requests to: Y.B. Ge, Deparment of Physiology, Nanjing MedicalUniversity, No 140, Hanzhong Road, 210029 Nanjing City, China. e-mail: ybge@njmu.edu.cn

DOI: 10.14670/HH-22.185

http://www.hh.um.es

Histology andHistopathologyCellular and Molecular Biology

air-conditioned and light controlled room at 24±2°C and60±5% humidity. After a 3-day adaptation period,animals were divided into six groups with differentfeeding periods of 1, 3, 7, 14, 28 days. Rats receiveddrinking water containing 6% (v/v) ethanol as their onlywater intake by the method of Turchan et al. (1999),control drinking water contained an equal caloricamount of dextrose. Water was offered ad libitum toanother group of animals for 28 days as the controlgroup. Each group contained 10 rats. At the end of eachfeeding period, the rats were anesthetized by urethaneand sacrificed. The stomachs were dissected and usedfor this study. Ethanol consumption averages 6.52 g/kgbody weight per day and the plasma ethanolconcentrations at time of stomach excision averaged18.47 mmol/L in this model. Dissociation of cells from stomach

The stomach cells dissociation was performed asdescribed by Zavros et al. (2000) with slightmodification. The stomachs were quickly excised fromseven rats in each group, washed in PBS (4°C), andtransferred to cold wash buffer. The stomach was openedalong the great curvature and rinsed with Hanks’balanced salt solution (Gibco BRL) with 0.1% bovineserum albumin (BSA). Dissociation of gastric cells wasperformed by incubating the whole stomach in 1%pronase (Merck USA) dissolved in 0.9% saline for 1.5 hrat 37°C. The solution was removed and replaced withfresh solution every 30 min. The cells were then treatedwith DNase I at 37°C for 30min, washed with coldHanks’ balanced salt solution with 0.1% BSA, andcentrifuged at 3000 rpm for 5 min at 4°C. The cells werefiltered through a 40-µm nylon cell mesh. Viability andyield were assessed by trypan blue exclusion.Approximately 5x106 cells in each rat were analyzed byflow cytometry.Flow cytometry

Aliquot cells (5x106 cells/tube) were washed in ice-cold PBS, fixed in ice-cold 70% ethanol and stored at4°C. They were then washed with PBS and treated withRNase (500/ml: Sigma) for 15 min at 37°C. Propidiumiodide (PI) (Sigma Chemical Co., Missouri) at 50µg/mlin PBS was used to stain DNA. Cell cycle analysis wasperformed using a Becton Dickinson fluorescence-activated cell analyzer (Becton Dickinson Co, CA) andthe Cellfit cell-cycle analysis software program providedby the manufacturer. Approximately 15000 cells werecounted for each determination. Histological and immunohistochemical analyses

The stomachs were excised from three rats in eachgroup and fixed in 10% neutral buffered formalin,embedded in paraffin and sectioned at 5µm for H&E andPCNA-cytokeratin 18 double staining. H&E staining

was performed according to the routine standardprocedures. The total cell number of each gastric glandwas counted from the uppermost parietal cell to thefundus of each gland of every 10th gland of the first 100glands viewed per slide for a total of 10 glands. Resultsare expressed as means of numbers±S.D. Slides wereexamined in a blinded fashion by coding them in a wayso the examiner was unfamiliar with the experimentalprotocol (Schmidt et al., 1985). PCNA- cytokeration 18double staining was performed according to themodified technique of Greenwell et al. (1991). Briefly,the sections deparaffinized in xylene, cleared in gradedethanol to PBS. Sections were then quenched in 3%hydrogen peroxide in 0.1% sodium azide to suppress theendogenous peroxidase activity and placed in an antigenretrieval solution consisting of 1% zinc sulfate indeionized water and irradiated for 7.5 min in a 700 Wmicrowave oven on full power. A routinestreptavidin–biotin protocol using the DAKO LSAB +Kits (HRP and AP) (Dako Japan, Kyoto, Japan) wasapplied. The tissue sections mounted on glass slideswere first incubated in 0.5% BSA in PBS to reducenonspecific protein binding, and then sequentiallyincubated to react with polyclonal anti-PCNA primaryantibody (NeoMarkers Lab Vision Corporation, CA)overnight at 4°C. The antibody was then linked withstreptavidin conjugated to horseradish peroxidase(HRP). HRP sites were visualized with 3,3’-diaminobenzidine (DAB) and H2O2. The slides wereimmersed three times with 0.1 M glycine-HCl buffer(pH 2.2) for 30 min. After washing with Milli-Q wateronce and with PBS three times, the sections were reactedwith monoclonal M30 CytoDEATH antibody (cloneM30; Roche, Mannheim, Germany) and AP-goat anti-mouse IgG successively, similar to that described above.The sections were visualized with nitroblue tetrazoliumchloride (NBT)/5-bromo-4-chlor-3-indolyl-phosphate(BCIP) in the dark. As a negative control, some sectionswere reacted with normal mouse IgG or normal rabbitIgG instead of the specific antibody.Quantitation of PCNA and M30 CytoDEATH labeling

An image analysis system (NYD100) was used forquantitative analysis of cell density (cell number/viewfield) of the PCNA-positive and M30 CytoDEATH-positive cells in the rat stomach. Three sections fromthree rats were used. PCNA-positive and M30CytoDEATH-positive cells were counted in fiverandomly selected view fields per section at amagnification of x400. At least 15 view fields in eachgroup were analyzed. Results are expressed as means ofnumbers±S.D. Statistical analysis

One-way analysis of variance (ANOVA) was used toestimate the significance of data obtained. P value lessthan 0.05 was considered to be significantly different.

186Gastric cell proliferation and apoptosis in chronic drinking rats

Student’s t-test was used for the comparison of databetween groups.Results

Flow cytometric analyses

Compared to the control groups, the percentage ofcells in S-phase was significantly increased in the ratstreated with 6% ethanol for 3, 7 and 14 days, anddecreased in the animals treated with ethanol for 28days. The percentage of apoptositic cells in gastricmucosa of rats was significantly enhanced duringexposure to the ethanol (data described in Table 1, Fig 1).Histological analyses

There was no inflammatory proliferative change inthe gastric mucosa in each group of rats treated withethanol, even those exposed to the ethanol for 28 days(Fig. 2A). The mean total cell number was described inTable 2. Immunohistochemical analyses

The positive PCNA-stained cells with uniformly

187Gastric cell proliferation and apoptosis in chronic drinking rats

Fig. 1. Typical flow cytometric analysis results of the dissociatedgastric cells in rats treated with ethanol: A, control; B, 2 weeks andC, 4 weeks. The histogram was constructed with at least 10000cells, and the result of the percent of S phase cells and apoptositiccells was described in Table 1.

Table 1. Changes in number of the cells in S phase and the apoptositiccells in Gastric mucosa of the chronic drinking rats.

Groups S phase cells Apoptositic cells

Controls 3.31±0.05 a 0.03±0.00 a1st 3.44±0.07 a 0.09±0.01 a3rd 5.16±0.10* a 0.40±0.05* a7th 6.53±0.04* a 0.43±0.07** a14th 7.84±0.09** a 0.60±0.05** a28th 2.40±0.01*a 0.33±0.04*a

*: P<0.05,**: P<0.01 VS Controls, a: P<0.05 seven samples were takenfrom each group and the number of cells in each group were at leastmore than 10000; (%,mean±SD, n=7).

Table 2. Mean total cell number per gastric gland in gastric mucosa ofthe chronic drinking rats.

Controls 1st 3rd 7th 14th 28th

54.8±0.5 56.1±0.5 55.9±0.1 56.2±0.4 55.7±0.5 50.1±0.3*

*: P<0.05, VS controls; (number/gland, mean±SD, n=5).

intense brown nuclei were located in the neck portion ofthe rat fundic gland in control rats, the position is thesame as reported by Yang et al. (1997) labeled gastricmucosa stem cells by Brdu. M30 CytoDEATH positivecells with intense blue cytoplasm were scattered in thesurface of the rat fundic gland (Figure 2-B).Significantly increased numbers of PCNA and M30CytoDEATH positive cells were observed in the fundicgland of the rats treated with the ethanol for 3 to 14days(Fig. 2C and Table 2). The decreased PCNA positivecells and increased M30 CytoDEATH positive cells weredetected in the gland of the rats treated with the ethanolfor 28 days (Fig. 2D and Table 3).Discussion

Cell proliferation can be measured by tritiated

thymidine incorporation followed by autoradiography(Langer et al., 1985; Eldridge et al., 1990; Lok et al.,1990), bromodeoxyuridine (BrdU) incorporationfollowed by anti-BrdU immunohistochemistry (Langeret al., 1985; Kamata et al., 1989; Eldridge et al., 1990;Arbern et al., 1994), flow cytometric cell cycle analysis(Langer et al., 1985; Lu et al., 1991; Beppu et al., 1994),and immunohistochemical labeling of proliferating cellnuclear antigen (PCNA) (Sarrat et al., 1991; Dietrich,1993; Beppu et al., 1994). All these methods can identifythe DNA synthesizing S-phase cells (Langer et al., 1985)in the cell cycle. The data obtained from the labeling ofS-phase cell in the cell cycle provide information aboutactivity of cell proliferation in the tissues. The level ofapoptosis was often assessed using propidium iodide(PI) staining (Koopman et al., 1994; Kieslinger et al.,2000), and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay,which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides (Nagarkatti, 2000;Moulian et al., 2001). Recently, a monoclonal antibodyby M30 CytoDEATH antibody directed against a neo-epitope of cytokeratin 18, which is formed after cleavageof this cytoskeletal protein by caspases, was shown to beof advantage over other tests for the detection of cellapoptosis (Leers et al., 1999; Austgulen et al., 2002;McPartland et al., 2005 ). The immunoreactivity of theM30 antibody is confined to the cytoplasm of apoptoticcells. The present study is designed to investigate, byimmunohistochemical labeling of PCNA and M30CytoDEATH combined flow cytometric cell cycleanalysis, the effect of ethanol on cell proliferation andapoptosis in the rats that received 6% ethanol for 28days, using an alcohol administration animal model,allowed to consume alcohol voluntarily. This method iscommonly used in the study of long-term alcohol intake(Turchan et al., 1999).

To our knowledge, the present study is the firstreport on the effect of long-term treatment with ethanolon cell proliferation and apoptosis in rat gastric mucosain vivo. The present study demonstrated a clear increasein the apoptosis cells in the stomach of animalssubjected to ethanol treatment for 3 to 28 days. Theproliferative cell numbers were increased in the ethanol

188Gastric cell proliferation and apoptosis in chronic drinking rats

Table 3. Numbers of the PCNA and M30 CytoDEATH positive cells inthe gastric Mucosa of the chronic drinking rats.

Groups PCNA cells M30 CytoDEATH cells

Controls 21.6±0.8 3.5±0.81st 19.8±0.5 4.3±0.63rd 34.9±1.0* 12.7±0.9*7th 42.3±0.7** 16.8±0.5**14th 39.7±0.8** 18.1±0.1**28th 4.0±0.5** 24.1±1.1**

*: P<0.05,**: P<0.01 VS Controls; (numbers/view field, mean±SD,n=15).

Fig. 2.A. Mucosa HE staining in rats exposed to the ethanol for 28 days.There was no inflammatory and proliferative change in the gastricmucosa. B-D. PCNA and M30 CytoDEATH immunohistochemicaldouble staining in the gastric mucosa of the chronic drinking rats by SP-POD and SP-AP methods respectively. B. Control animals, the M30CytoDEATH positive cells were scattered in the surface while the PCNApositive cells located at the neck portion in the fundic gland. C. Animalsexposed to the ethanol by 3 days, both of M30 CytoDEATH and PCNApositive cells increased in number. D. Animals exposed to the ethanolby 28 days, PCNA positive cells decreased while the M30 CytoDEATHpositive cells increased in number.

treated rats for 3 to14 days, and decreased at 28 days. Noinflammatory proliferative change was found in thegastric mucosa in each group of rats treated with ethanol,even those exposed to the ethanol for 28 days (Fig. 2A).These results suggested the enhancement of the cellturnover of gastric mucosa in the period of ethanoltreatment for 3 to 14 days, in which both cellproliferation and cell apoptosis is increased and the totalnumber of cells have no change in the ethanol treatedrats in this duration. These results also showed adecrease of cell turnover in the gastric mucosa of the ratstreated with the ethanol for 28 days by a reduction in cellproliferation and enhanced cell death by apoptosis and adecrease in the total number of cells was found in thegastric mucosa of these rats. The imbalance of cellproliferation and apoptosis has been implicated in long-term ethanol treatment.

The ability of the gastric mucosa to resist injury byendogenous secretions (acid, pepsin, and bile) and byingested irritants (e.g., alcohol, NSAIDs) can beattributed to a number of factors that have beencollectively referred to as “mucosal defense” (Wallaceand Granger, 1996). The gastric mucosal barrier is acomplex system made up of submucosal epithelial andmucus elements (Allen et al., 1993). Epithelial cellproliferation and migration leads to re-epithelializationof the ulcer crater, and the reconstruction anddifferentiation of glands. The functional integrity of thegastric mucosa and its secretory pit-gland units ismaintained through the constant renewal of theepithelium. The ongoing generation of new epithelialcells is a fundamental mechanism in mucosal protectionof the stomach, suggesting that undamaged epithelialrenewal function is required and coordination activitiesof cell proliferation and apoptosis would be necessaryfor normal progression of post-wounding epithelialrepair and successful mucosal recovery (Silen and Ito,1985). The current study clearly demonstrates thatethanol was involved in the regulation of in vivoregeneration of rat gastric epithelial cells. Increased cellproliferation of the stomach in short-term ethanolfeeding (3-14 days) is a regenerative change subsequentto ethanol-induced chronic mucosal injury (Hernandez-Munoz et al., 2000), and an adaptive cytoprotection ingastric mucosa was correlated with the enhanced cellturnover rate (data not shown). Long-term (28 days)stimulus with the low concentration (6%) ethanol mayreduce the cell turnover of the gastric mucosa anddecrease the capacity of regeneration, which leads to thepathogenesis of mucosal injuries such as ulcer andgastritis. This finding is in agreement with those ofClayman (Clayman and Moncada, 1968), who reportedthat atropic gastric mucosa has an increasedsusceptibility towards damaging effects.

Growth factors promote the proliferation ofepithelial cells and their migration over the ulcer crater(Tarnawski et al., 1992; Szabo et al., 1994; Li et al.,1999). Chronic ethanol treatment could cause analteration of epidermal growth factor receptor in the

stomach (Wang et al., 1996, 1997). Without furtherexamination we can not rule out the possibility that theincreased cell proliferation of the gastric mucosa is aresult of the changes of growth factors and theirreceptors. The ethanol apparently can directly stimulatecell apoptosis in the stomach of the rats because the M30CytoDEATH labeled cells were observed sporadically inthe control animals and diffusely in the rats treated withethanol for 3 to 28 days (Fig. 2B, C, D). Moreinvestigations are needed to delineate the specificintracellular signaling pathways involved in the ethanolmodulation of gastric epithelial proliferation andapoptosis. Moreover, further evaluation will be requiredto draw real clinical implication of long-term ethanoldrinking.

In conclusion, long-term chronic ethanol feeding (28days) resulted in decreases of cell proliferation andincreases of cell apoptosis. This impairment of the cellturnover function of gastric mucosa may be associatedwith a weakening of the cytoprotective function of thestomach and be related to ethanol-induced gastriclesions, which may be one of the mechanismsunderlying the gastric pathology associated with alcoholabuse.Acknowledgements. A part of this work was supported by ScientificResearch Foundation for the Returned Overseas Chinese Scholars,Satate Education Ministry, China (1999747).

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Accepted September 13, 2006

190Gastric cell proliferation and apoptosis in chronic drinking rats