Environmental Toxicology and Pharmacology 27 (2009) 200–205

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Environmental Toxicology and Pharmacology

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Nicotine withdrawal alleviates acetic acid-induced gastric injury in rats

Mustafa Deniza, Hasan Hüseyin Sahinb, Sema Tekinb, Melike Yesillerb, Bugra Agaoglub,Sule Cetinel c, Berrak C. Yegena,∗

a Marmara University, School of Medicine, Department of Physiology, Haydarpasa 34668, Istanbul, Turkeyb Marmara University, School of Medicine, Haydarpasa 34668, Istanbul, Turkeyc Marmara University, School of Medicine medical students„ Department of Histology & Embryology, Haydarpasa 34668, Istanbul, Turkey

a r t i c l e i n f o

Article history:Received 18 July 2008Received in revised form 10 October 2008Accepted 17 October 2008Available online 28 October 2008

Keywords:Gastric ulcerNicotineAnxiolyticNeutrophil accumulation

a b s t r a c t

Epidemiological and experimental studies have demonstrated that cigarette smoking intensifies gastriculceration. Although nicotine can act as an anxiolytic and antidepressant, its withdrawal may also leadto increased anxiety and depression. In order to associate the toxic actions of nicotine on gastric mucosawith alterations of anxiety level and to evaluate the impact of nicotine withdrawal on the anxiety level andthe severity of ulcer, an acetic acid-induced ulcer model was used. Male Sprague–Dawley rats were giveneither tap water or nicotine bitartarate (50 �g/ml in drinking water) for 15 days, while another group ofrats had 5 days of withdrawal following 10 days of nicotine treatment. Ulcer was induced by acetic acidon the 15th day of the treatments, and the rats were followed for 3 days until they were decapitated andthe gastric tissues were obtained. Using the hole-board test, basal anxiety levels measured on the firstday of the treatments were compared with the measurements made at the early and late phases of ulcerinduction. Chronic administration of nicotine did not have a potentiating effect on acetic acid-inducedgastric ulcer, since the gastric injury, as assessed by both macroscopic and microscopic evaluation andincreased gastric myeloperoxidase activity indicating neutrophil recruitment, was not exaggerated or

attenuated by nicotine intake. On the other hand, nicotine withdrawal attenuated gastric mucosal injury,despite an increased level of anxiety. Smoking cessation, which triggers the onset of depressive symptoms

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. Introduction

Tobacco smoking is one of the leading causes of death in bothhe developed and the developing countries (Peto et al., 2000). Therimary addictive substance in tobacco is nicotine, which is a water-oluble alkaloid rapidly absorbed through the respiratory tract,astrointestinal tract, skin and mucous membranes and mainlyetabolized in the liver (Ejaz and Lim, 2005). Epidemiological and

xperimental studies have demonstrated that cigarette smokingnd peptic ulcer diseases are closely associated (Kato and Nomura,992; Iwata et al., 1995), while smoking and stress work syner-istically to intensify gastric ulceration (Quan and Talley, 2002).owever, studies of smokers have shown that nicotine can relieve

tress and reduce anxiety (Gilbert et al., 1989), which is expected

o attenuate the gastric injury.

In experimental studies, it was demonstrated that chronic nico-ine administration worsens ethanol- and stress-induced gastric

ucosal injury (Hui et al., 1991; Wong et al., 2002). Nicotine,

∗ Corresponding author. Tel.: +90 216 414 4736; fax: +90 216 418 3327.E-mail address: byegen@marmara.edu.tr (B.C. Yegen).

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382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.etap.2008.10.006

has a worthwhile positive effect on the gastric mucosa.© 2008 Elsevier B.V. All rights reserved.

hich accumulates in gastric juice (Lindell et al., 1997), increasesarietal and chief cell mass and peak acid output (Guo et al.,986). The adverse effects of nicotine on gastric mucosa werettributed to vascular factors (Hui et al., 1991), decreased synthe-is of prostaglandins (Wong et al., 2002; Baumeister et al., 1995),uppression of nitric oxide release (Ma et al., 1999a), reductionn mucus content and glutathione level (Wong et al., 2002), andncrease in gastric polymorphonuclear neutrophil (PMN) recruit-

ent in the gastric tissue (Qui et al., 2004). Smoking also increasesile salt reflux rate and gastric bile salt concentration, thereby

ncreasing reflux-induced gastric ulceration (Maity et al., 2003).icotine plays an aggressive role in the development of gastro-uodenal lesions by elevating the endogenous vasopressin level,

ncreasing the production of platelet activating factor and endothe-in, and by reducing the level of circulating epidermal growthactor (Maity et al., 2003). The generation of reactive oxygen medi-tors (ROM) and ROM-mediated gastric mucosal cell apoptosis

s also involved in the aggravation of ulcer by nicotine (Ma etl., 1999b). Since these multi-systemic adverse effects of nicotinere withdrawn with the withdrawal of nicotine, it is expected toave reduced gastric injury. However, cessation of smoking typi-ally induces anxiety, irritability or impatience (Hatsumaki et al.,

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resulted in about a 60% reduction of the exploratory activity of therats, suggesting increased anxiety, as recorded by the decreasednumber of head-dipping activity on the second day of ulcer induc-tion (p < 0.001), when compared with the baseline measurements(Fig. 1). Nicotine administration, however, abolished the anxiogenic

M. Deniz et al. / Environmental Toxico

985) and potentiates stress-induced gastric damage (Wong et al.,002).

Although it is known that nicotine can act as an anxiolytic andntidepressant, it was also shown that adaptations to nicotine occurfter its chronic use, leading to increased anxiety and depressionollowing its withdrawal (Picciotto et al., 2002). In order to associatehe toxic actions of nicotine on gastric mucosa with alterations ofnxiety level and to evaluate the impact of nicotine withdrawal onhe anxiety level and the severity of ulcer, in the current study wesed acetic acid-induced ulcer model in rats with continuous oral

ntake of nicotine.

. Materials and methods

.1. Animals

Sprague–Dawley rats of both sexes (250–300 g) were kept at a constant temper-ture of 22 ± 2 ◦C with light–dark cycles of 12 h and fed a standard diet and waterd libitum. Studies were approved by the Marmara University Animal Care and Useommittee.

.2. Experimental design

Rats were randomly accustomed to four groups. Three groups had ulcer induc-ion with acetic acid and were followed for 3 days until they were decapitated.mong the ulcer groups, the rats in the nicotine-treated group (n = 8) were givenicotine bitartarate (50 �g/ml; Sigma, St. Louis, MO) to drink for 18 days, starting

rom 15 days prior to ulcer induction and the following 3 days, while the untreatedroup (n = 8) was allowed to drink tap water throughout the 18 days of the experi-ent. This concentration of nicotine was selected because it was previously shown

o produce a serum concentration of nicotine similar to that of a heavy cigarettemoker (Wong et al., 2002). In the third ulcer group that had nicotine withdrawaln = 8), rats were given nicotine in water (50 �g/ml) for 10 days, but tap water washen replaced for nicotine starting by the fifth day prior to ulcer induction and con-inued for the following 3 days. The rats in the fourth experimental group (n = 8,ontrol group) had sham operation without ulcer induction and received tap water.t the end of the 18-day follow-up period, all rats were decapitated and the stomachas opened along the greater curvature. Macroscopic ulcer index was calculated bylotting the injured areas on a transparent paper with 1 mm2 grids. Gastric tissueamples from each animal were fixed in 10% buffered p-formaldehyde and preparedor routine paraffin embedding. In addition, samples from each animal were stored at80 ◦C until the determination of tissue myeloperoxidase (MPO) activity, measureds an index of tissue neutrophil infiltration.

.3. Induction of gastric ulcer

Rats that were given either tap water or nicotine for 15 days and the rats that haddays of withdrawal following a 10 days of nicotine treatment were anesthetized

100 mg/kg ketamine and 0.75 mg/kg chlorpromazine; i.p.) after an overnight fast-ng. Ulcer was induced using a model modified from that originally described bykabe and Pfeiffer (1972). Briefly, midline laparotomy was performed and the stom-ch was gently exteriorized. Half a milliliter of acetic acid (HAc; 80%, v/v) was pouredhrough the barrel of a 3-ml syringe placed on the serosal surface of the stomach inhe corpus region and was allowed to remain in contact for 1 min. Then, the fluidas aspirated carefully and the area that remained in contact with the acid was gen-

ly rinsed with saline. The area exposed to HAc was nearly 60 mm2. The rats werellowed to recover from anesthesia and received normal chow and tap water (withr without nicotine) ad libitum for the following 3 days until they were decapitated.

.4. Evaluation of anxiety

It is well known that an increase in anxiety reduces exploratory behavior in rats,hich can be tested by using the hole-board test (Marco et al., 2005). The hole-

oard apparatus, providing a measure of direct exploration in rats (File and Wardil,975), consisted of a wooden board (55 cm × 65 cm × 47 cm) with 16 equally spacedoles (each 3.8 cm in diameter). The hole-board test was performed by placing theat in the center of the wooden board where the free movement of animals wasecorded by a video camera for 5 min. Then the number of head-dipping movementsbserved during the recording time, were counted from the videotape by a trainedbserver, who was blind to the treatment conditions (Boissier and Simon, 1962). Aecreased number of head-dipping indicated a reduction in the exploratory behavior

nd indicated increased anxiety.

The hole-board test was performed on the first day of the experimental proce-ures to record the basal values. Then the tests were repeated at 48 h after ulcer

nduction on the 17th day of the experiment. The number of head-dipping withespect to the initial record was expressed as a percentage of the control value (100%)or each animal.

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d Pharmacology 27 (2009) 200–205 201

.5. Tissue myeloperoxidase assay

Since MPO is released mainly by neutrophils in response to various stimula-ory substances, it is considered as an index for the evaluation of tissue neutrophilnfiltration (Nishida et al., 1996). Tissue-associated MPO activity was determinedn 0.2–0.3 g samples. The tissue samples were homogenized in 10 vol% of ice-coldotassium phosphate buffer (20 mM K2HPO4, pH 6.0). The homogenate was cen-rifuged at 12,000 rpm for 10 min at 4 ◦C, and the supernatant was discarded. Theellet was then rehomogenized with an equivalent volume of 50 mM K2HPO4 con-aining 0.5% (w/v) hexadecyltrimethylammonium hydroxide (Sigma). MPO activityas assessed by measuring the H2O2-dependent oxidation of o-dianisidine 2HCl.ne unit of enzyme activity is defined as the amount of the MPO present that causeschange in absorbance of 1.0 min−1 at 460 nm and 37 ◦C (Bradley et al., 1982).

.6. Microscopic evaluation

Tissue samples that were fixed with neutral-buffered formalin and embeddedn paraffin wax, were cut in 4-�m sections and stained with hematoxylin and eosinnd examined under an Olympus BX-51 photomicroscope. Histological assessmentsere made by an experienced histologist who was unaware of the treatments. Based

n the criteria outlined in the study by Gue et al. (1997), grading of the gastric injuryas made by giving the scores of 0–3 (0: none; 1: mild; 2: moderate; 3: severe)y evaluating the following criteria: ulceration, mucosal cell depletion, mucosaltrophy, edema, inflammatory cell infiltration and vascular dilatation.

.7. Statistics

All values are reported as means ± S.E.M. One-way analysis of variance withhe Newman–Keuls post hoc test was used to determine whether data of differ-nt groups were statistically different, where p < 0.05 was considered statisticallyignificant.

. Results

Ulcer induction in the group that was given normal tap water

ig. 1. The effect of nicotine and nicotine withdrawal on the anxiety levels (% head-ipping) of rats induced with acetic acid ulcer. A reduction in head-dipping indicates

ncreased anxiety. ***p < 0.001, compared to control group; +p < 0.05, compared to tapater given group.

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ffect of ulcer induction (p < 0.05). On the other hand, the rats withicotine deprivation had reduced head-dipping activity, as seen inhe tap water group, suggesting increased anxiety.

Ulcer index was increased in both tap water and nicotine receiv-ng ulcer groups, as compared to non-ulcer control group (p < 0.001;ig. 2A). On the other hand, nicotine withdrawal for 5 days beforend 3 days after ulcer induction significantly decreased (p < 0.05)he ulcer index. However, in all ulcer groups, which received

ither tap water or nicotine or which had nicotine withdrawal,icroscopic scores were increased with respect to control group

p < 0.001), and no significant difference was observed among thelcer groups (Fig. 2B).

ig. 2. The effect of nicotine and nicotine withdrawal on the (A) gastric ulcer indexnd (B) microscopic damage scores of rats induced with acetic acid ulcer. ***p < 0.001,ompared to control group; +p < 0.05, compared to tap water given ulcer group;p < 0.05, compared to nicotine-treated group.

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Microscopic analysis of the gastric tissue samples from thelcer group that received tap water presented with subepithelialasocongestion, swelling and spoiling of the epithelial cells, andncreased neutrophil infiltration in the lamina propria (Fig. 3B),

hile a regular epithelial line and lamina propria were observedn the control group (Fig. 3A). In the gastric tissues of nicotine-reated ulcer group, enlarged surface and pit epithelial cells wereoticed, with a high grade of PMN infiltration and vasocongestionFig. 3C). Similarly, nicotine-deprived ulcer group demonstratedevere epithelial damage with opened gastric glands, along withMN infiltration and vasocongestion (Fig. 3D).

MPO activity was significantly elevated in the gastric tissues oflcer-induced rats that received either tap water (p < 0.01) or nico-ine (p < 0.05) as compared to control group without ulcer (Fig. 4).imilar to the macroscopic damage scores, nicotine withdrawalrevented the ulcer-induced elevation in gastric MPO activityp < 0.01).

. Discussion

The results of the present study confirmed that chronic admin-stration of nicotine did not have a potentiating effect on aceticcid-induced gastric ulcer, since the gastric injury, as assessedy both macroscopic and microscopic evaluation and increasedastric MPO activity indicating neutrophil recruitment, was notxaggerated or attenuated by nicotine intake. However, nico-ine administration abolished ulcer-induced anxiety of rats. Thesendings suggest that the anxiolytic effect of nicotine does notlter the severity of gastric ulceration. Although the histologicalata demonstrating both epithelial and subepithelial damage wasot different between ulcer groups, nicotine withdrawal prior tolcer induction resulted in a decrease in macroscopically observeducosal damage along with an inhibition on neutrophil infiltra-

ion. Despite the ameliorating effect of nicotine withdrawal on thextent of gastric injury, cessation of nicotine intake increased thenxiety level back to those of saline-treated ulcer group, implicat-ng that its action on the gastric mucosa is not related with thetress-associated effects of nicotine.

Epidemiological data show that cigarette smoking increasesoth the incidence and relapse rate of peptic ulcer disease and alsoelays ulcer healing in humans. Previous studies have shown thathronic nicotine administration by itself does not cause mucosallceration, while it worsens ethanol-induced (Wong et al., 1986)nd stress-induced (Wong et al., 2002; Qui et al., 2004) gastricamage. In the current study, however, nicotine did not have andditive effect on acetic acid-induced gastric ulceration. This dif-erence may be due to the used ulcer model and the timing ofbservation following ulcer induction, which was in terms of hoursn the other ulcer models, in contrast to 4 days of post-ulcer periodn the current study, where the healing process may have started.

oreover, in different ulcer models, the involvement of aggressiver defensive mechanisms in the ulcerogenesis may be of varyingominance.

Nicotine has been shown to increase parietal cell mass andeak acid, which is suggested to be involved in the potentiationf secretagogue-induced gastric damage (Guo et al., 1986; Robert,972). While low doses of nicotine can trigger the stimulation ofNA synthesis and cell proliferation in vascular endothelial cells

Villablanca, 1998), it was shown to have adverse effects on wounde-epithelization (Zia et al., 2000), ulcer healing, cell proliferation

nd angiogenesis (Ma et al., 1999b, 2000). Smoking or nicotinedministration may result in abnormal blood supply to the tissuesy the inhibition of normal process of angiogenesis during woundealing, disturbing the microcirculation of the gastric mucosa (Mat al., 1999b), as well as the other tissues leading to decreased

M. Deniz et al. / Environmental Toxicology and Pharmacology 27 (2009) 200–205 203

Fig. 3. The micrographs of nicotine-treated and nicotine-withdrawn rats with acetic acid-induced gastric ulcer. (A) Control group, regular layout of superficial cells (*) withgastric neck cells (arrows), 200×, HE. (B) Ulcer group, subepithelial vasocongestion, swelliincreased accumulation of neutrophils in the lamina propria (arrow), 400×, HE. (C) Nicothigh grade of PMN infiltration (arrow) and vasocongestion, with less spoiled superficial esevere dilation of gastric glands (arrows), along with high PMN infiltration (arrowheads)

Fig. 4. The effect of nicotine and nicotine withdrawal on the gastric myelperoxidaseactivity (MPO) of rats induced with acetic acid ulcer. *p < 0.05 and **p < 0.01, com-pared to sham-operated control group; ++p < 0.01, compared to tap water given ulcergroup; &&p < 0.01, compared to nicotine-treated group.

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ng and destruction of the epithelial cells, dilation of gastric glands (arrowhead) andine-treated ulcer group, prominent dilation of surface and pit glands (arrowhead),pithelium (*), 200×, HE. (D) Nicotine-deprived ulcer group, epithelial damage withand vasocongestion in the lamina propria, 200×, HE.

epair and remodeling, which are common problems among smok-rs (Hashimoto et al., 2004; Ejaz and Lim, 2006). Nicotine does notnly enhance the gastric aggressive factors, but the defensive fac-ors are depressed by nicotine intake. In stressed animals, chronicicotine treatment dose-dependently decreases the thickness ofucus (Wong et al., 2002), which participates in the defense of

astric mucosa (Qui et al., 2004; Yeomans, 1988). Smoking reducesastric prostaglandin synthesis or release (McCready et al., 1985;uimby et al., 1986) and the circulating levels of epidermal growth

actor (Hashimoto et al., 2004), thereby making the mucosa sus-eptible to ulceration.

Studies on smokers have shown that nicotine can reduce anxietynd relieve stress (Picciotto et al., 2002), which may be responsibleor smoking more cigarettes when under stress. Based on anxietyxperiments in rodents, it was reported that nicotine can act asn anxiolytic and an antidepressant, while adaptation that occursy its chronic use may result in increased anxiety and depressionollowing withdrawal (Picciotto et al., 2002), which is found to bessociated with a significant decrease in brain reward thresholdEpping-Jordan et al., 1998). On the other hand, nicotine, like acutetress, is shown to stimulate the hypothalamic pituitary adrenalxis (HPA), leading to increases in levels of circulating corticos-eroids, in both animals and human smokers (Caggiula et al., 1991;

ilkins et al., 1982). The ability of nicotine to act as an anxiolyticr an anxiogenic is dependent on the anxiety model tested, theoute and time course of administration (Picciotto et al., 2002).n the current study, chronic pretreatment with nicotine relievedhe anxiety of animals that were stressed with the presence of

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astric ulcer. On the other hand, this anxiolytic effect of nicotineas lost when rats were deprived of nicotine for 5 days. Simi-

arly, it was shown that 2 days of nicotine withdrawal can increasenxiety in rats, suggesting that nicotine withdrawal can increaseepressive-like symptoms (Djuric et al., 1999). These studies sug-est that nicotine treatment alters the activity of HPA axis, whichight predispose the subjects to depressive episodes during with-

rawal due to reduced levels of corticosteroids (Picciotto et al.,002).

Smoking and stress also disturb the microcirculation of theucosa and have a synergistic negative effect on mucosal integrity

Ma et al., 1999a). In addition, stress lowers the amount of mucusdhering to gastric mucosa (Green et al., 1981), while tobaccomoke directly inhibits its synthesis (Yeomans, 1988). An earliereport by Qui et al. (2004) has demonstrated that nicotine inten-ified stress-induced gastric ulceration by a mechanism that wasnrelated to gastric mucosal neutrophil infiltration. Similarly, inhe present study, acetic acid-induced elevation in MPO activity,howing neutrophil infiltration, was not further increased in theastric tissues of rats with ulcer when they were administeredicotine as a pretreatment. However, nicotine withdrawal signifi-antly depressed MPO activity. It appears that the gastroprotectionchieved by nicotine withdrawal involves an inhibitory effectn tissue neutrophil infiltration that limits neutrophil-derivedxidative tissue damage. In concomitant with the MPO results,he macroscopic appearance of the mucosa was also improvedn the nicotine-withdrawn group, while vascular congestion anddema were still observed in histological evaluation. It is likelyhat the withdrawal of the adverse effects of nicotine has a

ore prominent impact on the mucosa than on the subepithelialissue.

Chronic intake of nicotine, despite its anxiolytic effect, does notlter the severity of acetic acid-induced gastric ulcer. On the otherand, upon the withdrawal of nicotine, the animals demonstraten increased level of anxiety, while mucosal injury is attenuated.lthough nicotine does not enhance ulcerogenesis in acetic acidlcer model, its withdrawal is a relief for the gastric mucosa, inde-endent of the mood of the animals. In conclusion, it should beoted that smoking cessation, which triggers the onset of depres-ive symptoms with nicotine withdrawal, still has a worthwhileositive effect on the gastric mucosa.

onflicts of interest

There is no financial disclosure or any funding source (excepthe general research funding supplied by the Marmara Universitychool of Medicine financial sources) in the study entitled “Nico-ine withdrawal alleviates acetic acid-induced gastric injury inats”.

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