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Exp Physiol 100.8 (2015) pp 881–895

Research Paper

Effects of different routes of nicotine administration on gastric morphology and hormonal secretion in rats Soad Shaker Ali1 , Enas Ahmed Hamed2 , Nasra Naeim Ayuob1,3 , Ahmed Shaker Ali4 and Mansour Ibrahem Suliman4 1

Anatomy Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia Physiology Department, Faculty of Medicine, Assuit University, Asyut, Egypt 3 Histology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt 4 Pharmacology Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

Experimental Physiology

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New Findings r What is the central question of this study? Does chronic administration of nicotine by different routes affect gastric hormonal secretions and morphology in rats? r What is the main finding and its importance? Chronic nicotine administration increased levels of gastrin, ghrelin and histamine but decreased prostaglandin E2 . Nicotine administered orally and by inhalation had a marked negative impact on the histological structure of the gastric mucosa compared with intraperitoneal administration. The negative impact of nicotine administration on gastric structure was associated with an increased concentration of gastrin and decreased prostaglandin E2 , which might be the cause of gastric/peptic ulcers in heavy smokers. The increase in ghrelin concentration and its effect following chronic nicotine administration needs further investigation.

The aim was to assess the effects of different routes of chronic nicotine administration on gastric morphology and hormonal secretion; mainly gastrin, ghrelin, histamine and prostaglandin E2 (PGE2 ). Forty adult male albino rats were randomly assigned into four groups (10 rats per group), treated for 21 days as follows: control group (given standard rat pellets and water only); oral nicotine-treated group [50 µg (ml drinking water)−1 ]; intraperitoneal nicotine-treated group [0.5 mg (kg body weight)−1 ]; and inhaled nicotine-treated group [0.5 mg (kg body weight)−1 ]. Concentrations of gastrin, ghrelin, PGE2 and histamine in serum and gastric tissue homogenates were assessed using ELISA kits. Stomach fundus was processed for histopathology and immunohistochemistry using light and electron microscopy. Different routes of chronic nicotine administration resulted in a significant increase in serum and gastric homogenate gastrin and ghrelin concentrations and a significant decrease in serum and homogenate PGE2 concentrations compared with the control group. Moreover, nicotine administration via oral and inhalation routes caused gastric erosion, transformation of peptic cells into the mucous variety, a significant increase in parietal cell numbers and an increase in expression of gastrin. In conclusion, the negative impact of nicotine administration on gastric structure that is associated with an increased concentration of gastrin and decreased concentration PGE2 might be the leading cause of gastric/peptic ulcers in heavy smokers. The increased ghrelin concentration and

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DOI: 10.1113/EP085015

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its effect following nicotine chronic administration needs further investigation. Based on these findings, we suggest that the alteration in gastric structure following chronic administration of nicotine can be prevented by reducing gastrin secretion and/or targeting its receptors. (Received 11 December 2014; accepted after revision 6 May 2015; first published online 16 June 2015) Corresponding author N. N. Ayoub: Department of Anatomy, Faculty of Medicine, King Abdulaziz University, P.O Box (80200), Jeddah 21589, Saudi Arabia. Email: [email protected] or [email protected]

Introduction Cigarette smoking continues to be the leading cause of preventable death and is the main risk factor for major diseases, such as chronic obstructive pulmonary disease (Kotz et al. 2014). Smokers who do not stop smoking lose at least one decade of life expectancy (Jha et al. 2013). Nicotine has been shown to delay ulcer healing in stress-induced, ethanol-induced or Helicobacter pylori-induced gastric injury in rats, as well as increased recurrence of duodenal ulcer (Zhang et al. 2009). The mechanisms by which cigarette smoking or nicotine adversely affect the gastric mucosa have not been fully elucidated. It has been suggested that cigarette smoke induces formation of ulcers and delays their healing through enhancement of acid and pepsin secretion, free radical production and infiltration of neutrophils (Chow et al. 1998). Reduction of gastric blood flow and secretion of mucus, prostaglandin (PG) and epidermal growth factor (Ma et al. 2000), as well as reduction of ornithine decarboxylase activity and polyamine synthesis (Shin et al. 2002), are other reported causes. It is known that the gastric hormone, gastrin, secreted by gastric (G) secreting cells in the stomach and TG cells in the duodenum, participates in the stimulation of gastric acid secretion (Green et al. 1989). In 1999, Kojima and colleagues identified a 28-amino-acid peptide, ghrelin, with an octanoyl reside at Ser3, that was found to stimulate the growth hormone secretagogue receptor (GHS-R), resulting in the release of growth hormone from somatotrophs in anterior pituitary. In the rat, ghrelin was found to be manufactured mainly by A-like cells in the parietal mucosa (Dornonville de la Cour et al. 2001). Ghrelin has been proposed to stimulate gastric motility and acid secretion (Masuda et al. 2000). In rats and mice, and perhaps in other species, the ability of ghrelin to increase gastric motility appears to be dependent on both vagal and intrinsic neurons (Fukuda et al. 2004). Previous studies have demonstrated that ghrelin secretion is influenced by many gastrointestinal hormones, such as glucagon, insulin, cholecystokinin, glucagon-like peptide, gastrin and somatostatin (Lippl et al. 2004; Hosoda & Kangawa, 2007; Katayama et al. 2007). Although numerous studies in man and laboratory animals have examined the influence of nicotine on gastric

acid secretion, the effects of nicotine on gastrointestinal hormones that influence acid secretion have not been examined in depth. The purpose of this experimental study is to examine the effects of different routes of chronic nicotine administration on gastric morphology and hormonal secretion; mainly gastrin, ghrelin, histamine and prostaglandin E2 (PGE2 ). Methods Chemicals

Nicotine tartrate was purchased from Fluka AG (Chemische Fabric, Buch, Switzerland). Hydrogen tartrate salt of nicotine (Sigma N-5260) was dissolved in 0.9% NaCl solution to obtain a 0.15 mg ml−1 concentration of nicotine. The pH of the nicotine solution was adjusted to 7.4 using 0.1 N NaOH.

Experimental animals

A total of 40 adult male Wistar albino rats (weight 200–250 g, aged 3 months) were purchased from the animal house in King Fahd medical research centre. The rats were housed in wire-meshed cages at 24◦ C with constant humidity and 12 h–12 h light–dark cycle (light on at 05.00 h). The animals were fed ad libitum with a commercial rat diet consisting of pellets and tap water prior to the studies. All rats were acclimated for 2 weeks before the experimental protocol was started. All procedures described in the present study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National Academies Press (US); 2011) and were approved by the Biomedical Research Ethics Committee at the Faculty of Medicine, King Abdul Aziz University, Jeddah, Saudi Arabia. The rats were randomly assigned into four groups (10 rats per group), as follows: the control group was given standard rat pellets and water only; the oral nicotine-treated group was given nicotine tartrate orally in drinking water at a dose of 50 μg ml−1 for 21 consecutive days (Wong et al. 2002); the intraperitoneal nicotine-treated group was given I.P. nicotine tartrate injection dissolved in 0.9% normal saline once daily at

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Nicotine administration and gastric hormones secretion and structure

a dose of 0.5 mg (kg body weight)−1 (0.67–0.83 ml of the solution contained 0.15 mg nicotine ml−1 according to animal weight) for 21 consecutive days (Bartolini et al. 2011); and the inhaled nicotine-treated group was exposed to nicotine by inhalation, where conscious rats were placed in an exposure container (250 ml) for 1 h day−1 and exposed to a diluted mainstream of nicotine vapour dissolved in saline [5 mg (kg body weight)−1 ] for 21 consecutive days (Abobo et al. 2012) using an aerosol delivery system machine (Buxco Research Systems, Wimington, NC, USA).

Collection of samples

For all groups, 24 h after the last dose of nicotine, rats were anaesthetized by I.P. injection of phenobarbitone [Abbott Diagnostics, Cairo, Egypt; 50 mg (kg body weight)−1 ]. Deep anaesthesia was ensured after the rat lost the corneal reflex and all muscles became relaxed. The blood samples were then individually collected from retro-orbital plexus of veins because they are easily accessed and they provide a large amount of blood. The blood samples were centrifuged for 10 min at 600 G. and the sera were divided into small aliquots and stored at −80°C until use. Immediately after the blood specimens were taken, the rats were killed by decapitation, the abdomen was opened and the stomach tied at oesophageal and duodenal connections, perfused with cold saline, cut at the two ends and moved into a deep Petri dish containing saline. Ten minutes later, the stomach was opened through the greater curvature. The incised stomach was rinsed twice with ice-cold normal saline, examined macroscopically and photographed. The stomach was cut into two halves. The first half was cut into longitudinal strips; some strips were fixed in 10% neutral buffered formalin and processed to obtain paraffin blocks. Paraffin sections 5 μm thick were prepared and stained with Haematoxylin and Eosin or combined periodic acid–Schiff and Alcian Blue (PAS–Alcian Blue) to stain mucus-secreting cells and were visualized under the light microscope (Bancroft & Gamble, 2008). Other strips were fixed in 3% glutaraldehyde in sodium cacodylate buffer (pH 7.4) to be processed for visualization under both transmission and scanning electron microscope at the electron microscope unit, Assiut University, Egypt. The second half of the stomach was used to obtain gastric homogenates. Gastric mucosal proteins were extracted by boiling aqueous homogenates (1:10, tissue weight:water volume) for 20 min immediately after the dissection of tissue. The homogenates were kept on ice and centrifuged. Supernatants were lyophilized and suspended in appropriate assay buffers, and the samples were stored at −80°C until assay.

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Immunohistochemistry

Immunohistochemical staining for gastrin was carried out using the peroxidase-labelled streptavidin–biotin technique according to Ramos-Vara et al. (2008). Paraffin sections of gastric mucosa were deparaffinized, rehydrated in distilled water, treated in 3% H2 O2 for 5 min and rinsed with PBS for 15 min. Sections were incubated in blocking buffer (1.5% normal goat serum in PBS) for 30 min, then incubated for 45 min at room temperature with the primary antibody against gastrin (1:800 dilution; [A 0568; Dako Cytomation, Glostrup, Denmark), washed thoroughly with PBS and then incubated with a secondary biotinylated goat anti-rabbit IgG for 1 h, at room temperature. After thorough washing with PBS, the reaction products were visualized by immersing the section into the chromogen diaminobenzidine. Sections were counterstained with Haematoxylin, dehydrated and coverslipped. For the negative control specimen, the same steps were followed, but the primary antibody was omitted and replaced by PBS only. Computerized image analysis system software (Pro Plus image analysis software version 6.0 connected to an Olympus Microscope BX-51 with a digital camera connected to a computer at the microscope centre; King Fahd Center for Medical Research) was used for imaging as well as taking the morphometric measurements. The mean area percentage (AP) and mean intensity (MI) of gastrin were measured using an objective lens of ×20 magnification. Five random sections from each animal were examined and five readings from each animal were collected (Abd El-Haleem et al. 2013).

Hormonal measurements

The concentrations of gastrin, ghrelin, PGE2 and histamine in serum as well as in the supernatant of gastric tissue homogenates were measured using commercially available ELISA kits (gastrin, Ray Biotech, Inc., Norcross, GA, USA; and PGE2 , ghrelin and histamine, Glory Science Co., Ltd, Del Rio, TX, USA). Each assay was performed in duplicate according to the manufacturer’s instructions. The minimal detection limits for gastrin, PGE2 , ghrelin and histamine were 9.92 pg ml−1 , < 0.24 ng l−1 , < 0.24 pg ml−1 and < 0.54 ng ml−1 , respectively.

Statistical analysis

All data are expressed as means ± SD, and differences between the control and treated groups were determined by one-way ANOVA using SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). A value of P < 0.05 was considered significant.

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Results Hormone concentrations in serum samples and gastric homogenates

Gastrin concentrations in serum samples and gastric homogenates were significantly higher in oral and I.P. nicotine-treated groups compared with the control group (P = 0.001). The serum gastrin concentration was significantly higher in rats that received I.P. nicotine compared with the inhaled nicotine group (P = 0.040). However, the homogenate gastrin concentration was significantly higher in rats that received oral and I.P. nicotine compared with the inhaled nicotine group (P = 0.050 and P = 0.040, respectively). The ghrelin concentrations in serum samples as well as in gastric homogenates were significantly higher in oral, inhaled and intraperitoneal nicotine-treated groups compared with the control group (P = 0.001, P = 0.001 and P = 0.001, respectively, in serum samples; and P = 0.001, P = 0.001 and P = 0.010, respectively, in homogenate samples). The PGE2 concentrations in serum as well as in gastric homogenates were significantly lower in rats that received nicotine by inhalation, oral and I.P. routes compared with the control group (P = 0.001, P = 0.002 and P = 0.002, respectively, in serum samples; and P = 0.001, P = 0.001 and P = 0.001, respectively, in homogenate samples). However, the serum PGE2 concentration was significantly lower in rats that received nicotine by inhalation compared with rats that received oral and intraperitoneal nicotine (P = 0.010). Although there was an insignificant difference in the PGE2 concentration in gastric homogenates between all treated groups, the lowest concentration of PGE2 was found in the gastric homogenates extracted from the rats that received nicotine by inhalation. The histamine concentration in gastric homogenates was significantly higher in rats that received nicotine by the inhalation and oral routes compared with the control group (P = 0.010; Table 1).

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and inflammatory cell infiltrates were observed in the stomach of rats treated with nicotine compared with the control animals. Among the observed changes was the abnormal appearance of mucus-secreting cells in the lower parts of the glands; meanwhile, in control animals the mucus-secreting cells appeared only in the upper part of the gland. Moreover, there was a significant increase in the number of parietal cells in rats that received oral or I.P. nicotine compared with the control animals (P = 0.005 and P = 0.006, respectively; Fig. 2A). Examination of the gastric mucosal surface by scanning electron microscope revealed the presence of gastric erosions in rats that received oral or I.P. nicotine compared with the control group. Such erosions were accompanied by loss of surface cells and accumulation of cellular debris within the lumina of gastric glands. These changes were less frequently observed in animals exposed to nicotine by inhalation (Figs 1–4). In addition, examination of the gastric mucosa under the transmission electron microscope showed dilatation of cisterns of the smooth endoplasmic reticulum (sER) of the parietal cells in animals that received nicotine by all routes compared with the control group. Some peptic cells in the examined sections dissected from rats that received oral and I.P. nicotine showed transformation of the zymogen granules into the mucous variety (Figs 1–4). Immunohistochemistry

Immunostaining of gastrin showed upregulation of its expression by G cells in rats that received oral and I.P. nicotine compared with the control group (Figs 1E, 3F and 4E). This was confirmed quantitatively by measuring the mean intensity and area percentage of gastrin expression. In rats that received oral or I.P. nicotine injection, we found a significant increase in the mean intensity of gastrin expression (P = 0.002 and P = 0.006, respectively) and in the area percentage of gastrin expression (P = 0.004 and P = 0.001, respectively) compared with the control animals (Fig. 2A–C). Discussion

Macroscopic and microscopic examination of gastric mucosa

Macroscopic examination of the stomach of nicotine-treated rats revealed gastric mucosal hyperaemia and ulceration. These changes were prominent in oral nicotine-treated rats compared with inhalation and I.P. nicotine-treated rats (Figs 1–4). Light microscopic examination of gastric sections stained with Haematoxylin and Eosin showed various degrees of mucosal ulceration due to loss of surface mucous lining cells, which was confirmed by PAS–Alcian Blue staining (Figs 1–5). Small haemorrhagic lesions

In this study, we showed that different routes of nicotine administration not only had a negative effects on the morphology of the gastric mucosa but also had an impact on the functional activity of different gastric cell types, which was reflected by changes in the concentrations of gastrin, ghrelin, PGE2 and histamine in both serum samples and gastric homogenates. The serum gastrin concentration was significantly higher in animal groups that received nicotine via oral, inhalation or I.P. routes; the effects of I.P. nicotine injection could be attributed to stimulation of gastrin-secreting cells within and outside the stomach, e.g. TG cells of the duodenum. Moreover,  C 2015 The Authors. Experimental Physiology  C 2015 The Physiological Society

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Table 1. Effect of nicotine administered by different routes on gastrointestinal hormones Groups receiving nicotine Variable

Control (n = 10)

Serum gastrin (pg ml−1 )

108.7 ± 10.5

Homogenate gastrin (pg ml−1 )

150.4 ± 5.3

Serum ghrelin (ng ml−1 )

1.22 ± 0.2

Homogenate ghrelin (ng ml−1 )

1.2 ± 0.1

Serum prostaglandin E2 (ng ml−1 )

10.5 ± 1.8

Homogenate prostaglandin E2 (ng ml−1 )

16.9 ± 5.4

Serum histamine (ng ml−1 )

84.3 ± 6.7

Homogenate histamine (ng ml−1 )

57.8 ± 10.2

Oral (n = 10)

Inhalation (n = 10)

Intraperitoneal (n = 10)

714.5 ± 322.5 = 0.001

552.2 ± 212.2 ∗ P = 0.001 † P = 0.060

728.4 ± 268.1 ∗ P = 0.001 † P = 0.040 ‡ P = 0.880

746.7 ± 133.6 = 0.001

863.2 ± 168.3 ∗ P = 0.001 † P = 0.050

747.5 ± 205.3 ∗ P = 0.001 † P = 0.040 ‡ P = 0.990

2.20 ± 0.3 = 0.001

2.3 ± 0.6 ∗ P = 0.001 † P = 0.490

2.4 ± 0.6 ∗ P = 0.001 † P = 0.870 ‡ P = 0.440

1.9 ± 0.5 = 0.001

1.8 ± 0.6 ∗ P = 0.001 † P = 0.410

1.7 ± 0.7 ∗ P = 0.010 † P = 0.570 ‡ P = 0.220

8.6 ± 0.9 = 0.002

7.0 ± 1.6 ∗ P = 0.001 † P = 0.010

8.5 ± 1.1 ∗ P = 0.002 † P = 0.010 ‡ P = 0.930

4.9 ± 2.1 = 0.001

5.7 ± 2.6 ∗ P = 0.001 † P = 0.600

6.6 ± 3.0 ∗ P = 0.001 † P = 0.560 ‡ P = 0.320

84.1 ± 11.6 = 0.730

83.4 ± 9.5 ∗ P = 0.970 † P = 0.710

85.5 ± 5.2 ∗ P = 0.700 † P = 0.730 ‡ P = 0.520

74.7 ± 11.4 = 0.010

72.9 ± 24.3 ∗ P = 0.010 † P = 0.780

64.3 ± 12.9 ∗ P = 0.320 † P = 0.190 ‡ P = 0.160

∗P

∗P

∗P

∗P

∗P

∗P

∗P

∗P

Total (n = 30) 646.2 ± 26.0 = 0.001

∗P

798.7 ± 176.0 = 0.001

∗P

2.3 ± 0.5 = 0.001

∗P

1.8 ± 0.6 = 0.001

∗P

7.9 ± 1.5 = 0.001

∗P

5.7 ± 2.6 = 0.001

∗P

84.3 ± 9.0 = 0.970

∗P

71 ± 18.6 = 0.010

∗P

∗Significance versus control †significance versus oral; and ‡significance versus inhalation. Data are expressed as means ± SD. Comparisons between different groups were made using one-way ANOVA.

stomach gastrin expression was upregulated in both oral and inhalation groups compared with the control group. It is worth mentioning that the nicotine-induced changes in gastrin homeostasis may be attributed, in part, to nicotine-stimulated elevations in circulating adrenal catecholamine concentrations due to stress, as was reported by other researchers (Harty et al. 1988). The findings of the present study appeared to be in contrast to some previous studies that reported reduced serum and gastric levels of gastrin, which was  C 2015 The Authors. Experimental Physiology  C 2015 The Physiological Society

attributed, in part, to a nicotine-induced elevation in acid secretion. Gomez and colleagues (1996) found that nicotine administration in rats downregulated gastrin expression by G cells and reduced the serum concentration of gastrin because of increased gastric acid secretion. Meanwhile, Wong & Ogle (1995) reported that nicotine at a dose of 25 μg ml−1 in the drinking water given for 10, 30 and 45 days caused insignificant changes in either serum gastrin concentrations or in the parietal cell population, mucosal surface area and/or mucosal

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thickness in rats, and these insignificant changes could be attributed to the dilution effect of the drinking water. Gomez et al. (1997) reported that I.V. nicotine treatment alone (12 mg kg−1 day−1 for 14 days) did not affect serum

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gastrin levels, in rats which could be explained by the short duration of exposure. It was reported that gastrin induced gastric acid secretion from parietal cells of the stomach by stimulation of histamine release (Cui & Waldrum, 2007).

Figure 1. Control gastric mucosa Gastric mucosa of a control rat (A), showing normal appearance. B and C, fundic glands of control rat stomach, showing intact surface mucus-secreting cells (white arrow), intact parietal cells (thin black arrows) and pepsinogen-secreting cells (thick black arrow) in the upper (B) and basal parts of the glands (C) (Haematoxylin and Eosin, ×400 magnification). D, surface cells (dashed arrows) show mucopolysaccharides (periodic acid–Schiff reaction, ×200 magnification). E, immunoexpression of gastrin in fundic mucosa (immunohistochemistry, ×600 magnification). F, fundic mucosa, showing mucous surface cells with intact surface apices (black arrows), gastric pits (white thick arrows) and thin threads of mucus (white dashed arrows; scanning electron microscopy). G, parietal cell shows euchromatic nucleus (N), intact mitochondria (M) and smooth endoplasmic reticulum (sER; transmission electron microscopy). H, two adjacent peptic cells (P) with intervening enteroendocrine cell (E). The peptic cells show euchromatic nuclei (N) and intact cisterns of rough endoplasmic reticulum (rER; transmission electron microscopy).

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In addition, the results of the present study showed that chronic nicotine administration significantly increased the serum and gastric homogenate concentrations of ghrelin compared with the control group. These findings were supported by the study of Fagerberg et al. (2003), who reported that in 58-year-old Swedish men, current smokers had higher plasma ghrelin concentrations than non-smokers. Also, Lee et al. (2006) reported that ghrelin plasma concentration decreased with smoking cessation. Paslakis et al. (2014) found that ghrelin plasma concentrations in young adults who had been exposed to cigarette smoke in utero were significantly higher than in those without prenatal smoke exposure. In contrast, Mutschler et al. (2012) reported insignificance differences in ghrelin concentrations between smokers and non-smokers. However, their findings should be interpreted with caution because of the small sample size (11 male heavy smokers) and thus limited statistical power. As reported in previous studies, increased ghrelin secretion could also have an indirect influence mediated by other factors, including growth hormone (Wilkins et al. 1982), leptin (inverse correlation with ghrelin concentrations; Chan et al. 2004) and vagal nerve stimulation (Niedermaier et al. 1993).

Our findings suggested that smoking leads to release of intracellular ghrelin into the circulation. Fukumoto and coworkers (2008) suggested that gastrin induced gastric acid secretion by acting on X/A-like ghrelin cells, leading to ghrelin release. Thus, the elevated concentration of ghrelin that was found in the present study resulted either directly from the nicotine itself or indirectly from an increase in gastrin concentration after nicotine administration. The effect of ghrelin on the gastrointestinal tract concerns not only regulation of motility and secretion, but also participation in processes protecting against ulcerogenic factors. Previous studies had shown that ghrelin given peripherally (Sibilia et al. 2003; Brzozowski et al. 2004; Konturek et al. 2004; I´ s¸eri et al. 2005) or centrally (Sibilia et al. 2008) protected the gastric mucosa against various noxious agents. Pretreatment with ghrelin reduced gastric mucosal damage induced by ethanol (Sibilia et al. 2003; Konturek et al. 2004), stress (Brzozowski et al. 2004) or alendronate (I´ s¸eri et al. 2005) and accelerated the healing of gastric ulcers induced by acetic acid (Ceranowicz et al. 2009) and duodenal ulcers evoked by acetic acid or cysteamine (Ceranowicz et al. 2009; Warzecha et al. 2012). These observations suggested that the therapeutic effect of ghrelin in gastric ulcers had

A Mean number of parietal cells in gastric glands

120 p=0.06

100

p=0.06 p=0.05

80 60 40 20 0

Control

Oral Nicotine

p=0.09

180 160 140 120 100 80 60 40 20 0

IP Nicotine

130

p=0.003

p=0.07

110

p=0.002

90 Area %

Mean intenstiy (lum)

B

Inhalation Nicotine

p=0.001 p=0.004

70 50 30 10

Control

Oral Nicotine

Inhalation Nicotine

IP Nicotine

−10

Control

Oral Nicotine

Inhalation Nicotine

Figure 2. The mean number of partial cells in gastric glands (A), mean intensity (B) and percentage area of gastrin immunoexpression (C) in gastric glands of the studied group

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IP Nicotine

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a universal nature and was not dependent on the primary cause of gastric damage. The protective effect of ghrelin is related to factors with documented gastroprotective properties, including nitric oxide, PGs and the neurotransmitters of sensory afferent fibres (Konturek

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et al. 2006). Moreover, ghrelin, which was proposed to act via vagal stimulation, exerted anti-inflammatory properties because it reduced the expression of the pro-inflammatory cytokines interleukin-1β and tumour necrosis factor-α in gastric mucosa exposed to stress or

Figure 3. Gastric mucosa after oral nicotine administration Gastric mucosa after oral nicotine administration (A) shows hyperaemia and ulceration. It shows an erosion (dotted circles in B), and higher magnification (C and D) shows destruction (thin black arrows) and shedding (star) of the surface mucous cells and an increased number of oxyntic cells (white arrows) and some mucous cells (thick white arrows) in the lower part of the glands (B, C and D, Haematoxylin and Eosin, ×100, ×200 and ×400 magnification, respectively). E, mucus-secreting cells are absent in the ulcerated area (periodic acid–Schiff, ×100 magnification). F, an increase in the number of gastrin-positive cells is observed (immunohistochemistry, ×600 magnification). G, marked loss of surface cells, exposing the gastric gland opening (black arrows), cell debris and necrotic tissue are observed (white arrows; scanning electron microscopy). H, oxyntic cell shows dilated cisterns of smooth endoplasmic reticulum (sER) and cytoplasmic vacuoles (V; transmission electron microscopy). I, peptic cell (P) shows multiple apical mucous secretory granules (M; transmission electron microscopy). Abbreviation: rER, rough endoplasmic reticulum.

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ischaemia–reperfusion (Brzozowski et al. 2006; Konturek et al. 2006; Szlachcic et al. 2010; Gil et al. 2011). In rats treated with ghrelin, improvement of mucosal blood flow could result from increased perfusion through existing vessels and/or might be due the stimulation of angiogenesis (Ahluwalia et al. 2009). Warzecha et al.

(2014) revealed that ghrelin exhibits a therapeutic effect in the healing of experimental gastric ulcers induced by ethanol in rats. This effect seems to be related to the improvement of mucosal blood flow and mucosal cell vitality, an increase in mucosal cell proliferation and a reduction in local inflammation. Meanwhile, recent

Figure 4. Gastric mucosa after administration of nicotine by inhalation Gastric mucosa (A) after nicotine inhalation shows ulceration (black arrows). It shows haemorrhages (arrowhead) and cellular infiltrate (thick black arrow) between the fundic glands (B), and high magnification (C) shows loss of the surface cells (thin arrows; B and C, Haematoxylin and Eosin ×200 and ×400 magnification, respectively), which are mostly mucus-secreting cells (dashed arrows in D; periodic acid–Schiff, ×100 magnification). E, an increase in the number of gastrin-positive cells is observed (immunohistochemistry, ×600 magnification). F, slight degeneration of the apical surface of gastric mucous cells (black arrows) is seen (scanning electron microscopy). G, an oxyntic cell shows a euchromatic nucleus (N), intact mitochondria (M) and slightly dilated smooth endoplasmic reticulum (sER) cisterns (transmission electron microscopy). H, peptic cells (pc) with dilated cisterns of rough endoplasmic reticulum (transmission electron microscopy).

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reports have revealed that ghrelin is overexpressed in multiple malignant carcinoma cells (Gahete et al. 2011; Majchrzak et al. 2012) and plays important roles in tumorigenesis (Waseem et al. 2008). Ghrelin may modify the expression of gastrin in gastric mucosa (An et al. 2007), leading to carcinogenesis (Nikolopoulos et al.

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2010). Tian and colleagues (2013) suggested that ghrelin promoted cell proliferation, migration and invasion via the activation of the nuclear factor-κB/P65 signalling pathway in gastric cancer. Ghrelin/GHS-R/nuclear factor-κB signalling promoted cell proliferation by accelerating the G1- to S-phase transition of gastric cancer cells. Similar

Figure 5. Gastric mucosa after intraperitoneal nicotine administration Gastric mucosa (A) after I.P. nicotine administration shows ulcerated mucosa (black arrows). It shows a deep gastric ulcer (dashed arrows in B), and higher magnification views (C and D) show nuclear and cellular debris (white star), small haemorrhages (white arrows), hyperplasia and enlargement of oxyntic cells (thin black arrows) and a reduced number of basophilic peptic cells (thick black arrows; B, C and D, Haematoxylin and Eosin, ×100, ×400 and ×600 magnification, respectively). Loss of mucus-secreting cells in some areas (E) (dashed arrows; periodic acid–Schiff, ×100 magnification) and an increased number of gastrin-positivve cells (F) are observed (immunohistochemistry, ×600 magnification). G, some surface cells are lost (black arrows), exposing the gastric gland openings (white arrows; scanning electron microscopy). H, oxyntic cell shows dilated smooth endoplasmic reticulum (sER) cisterns and swollen mitochondria (arrows; transmission electron microscopy). I, peptic cells (P) with some dilated cisterns of rER and mucous transformation (M) are seen (transmission electron microscopy). Abbreviation: rER, rough endoplasmic reticulum.

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to the results in cell experiments, ghrelin/GHS-R/P65 signalling could promote gastric cancer growth in vivo. Moreover, Tian and colleagues (2013) also found that ghrelin/GHS-R/P65 signalling elevated the mRNA and protein expression of the tumour metastasis marker matrix metalloproteinase-2, and promoted gastric cancer cell migration and invasion. Thus, the role of ghrelin in peptic ulceration induced by nicotine is protective or carcinogenic requires further molecular investigations. In the present study, we also found that PEG2 concentrations in both serum and gastric homogenate were significantly decreased in nicotine-treated animals compared with the control rats. This reduction was significant in serum concentrations of PEG2 in rats that inhaled nicotine compared with those that received oral or I.P. nicotine; meanwhile, there was an insignificant difference in the concentration of PEG2 in homogenate of the rats that received oral nicotine compared with those that received nicotine via inhalation and I.P. injection. This might indicate that the histopathological changes observed in the groups that received nicotine could be attributed directly to the concentration of PEG2 in homogenate rather than the serum concentration. The oral bioavailability of nicotine was reported to be low (20–45%) compared with other routes, and these pharmacokinetic differences may explain the variability in reduction of PG concentrations (Hukkanen et al. 2005). These findings were supported by other researchers, who reported that PGE2 synthesis in the gastric mucosa was decreased in smokers (Baumeister et al. 1995). Prostaglandins, especially PGE2 , have cytoprotective effects on gastric mucosa as a consequence of various physiological mechanisms, including increased epithelial mucus and bicarbonate secretion, as well as inhibition of free radical activities and enzymes released from neutrophils (Morris et al. 1998; Igarashi et al. 2014), and these finding could account for the histopathological changes induced by nicotine in the gastric mucosa. Histamine plays a pivotal role in the regulation of various gastric functions, such as acid secretion, motility and mucosal blood flow. Histamine increased gastric mucosal permeability to electrolytes and rendered the mucosa more susceptible to acid-induced damage (Hung, 2001). The results obtained in the present study revealed that serum histamine did not show any significant changes in rats that received nicotine by any used route, while homogenate histamine showed a significant increase in rats that received nicotine via oral and inhalation methods. Therefore, the results of this study suggest that systemic gastrin and/or ghrelin are more involved, rather than a histaminergic mechanism, in inducing changes in the gastric mucosa after chronic nicotine administration. This view is supported by the finding that the hypersecretion of histamine is  C 2015 The Authors. Experimental Physiology  C 2015 The Physiological Society

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accompanied by an increased mobilization of gastric histamine reflected by increase in histamine concentration in gastric homogenate. Cigarette smoke and nicotine stimulate H1 and H2 receptors in the stomach (Chow et al. 1998), probably by stimulating mast cell degranulation to release more histamine (Ogle et al. 1993). In this respect, Hladovec (1978) investigated the release of histamine during inhalation of cigarette smoke and anoxaemia in the heart–lung and intact dog preparation. They suggested that there was a definite release of histamine during the period of anoxia and that the initial bronchoconstriction associated with inhalation of cigarette smoke might induce a localized state of anoxia that leads to release of histamine. We also found that nicotine induced gastric mucosal hyperaemia, ulceration and small haemorrhages. These findings were in line with the study by Hui et al. (1991), which showed that oral nicotine administration (25 μg ml−1 ) for 9 days resulted in microscopic injury of the gastric mucosa. Using a light microscope, gastric gland of the rats that received nicotine by different routes showed an increased number of parietal cells in addition to signs of parietal cell hyperactivity, e.g. dilatation of cisterns of the sER observed by electron microscopy. It seems that the increased gastrin concentrations in serum and gastric homogenate, documented in the present study, as well as the increase gastrin expression underlie the hyperactivity of parietal cells and result in increased stomach acidity. Transformation of peptic cells into mucous-like cells in the lower part of the gastric glands after chronic nicotine administration could be, in part, a compensatory protective defense in response to the increase in gastric parietal cell numbers and secretion. Gastric and intestinal metaplasia upon nicotine administration was recently reported by Morais et al. (2014). The significant increase in gastrin concentrations in nicotine-treated animals might explain the gastric erosions that have been seen upon macroscopic examination of the stomach of nicotine-treated rats, because gastrin is an important stimulator to gastric acid secretion. Moreover, the decrease in PGE2 concentration in nicotine-treated animals compared with the control group might be an additional cause for development of gastric erosions. Prostaglandin E2 is an important regulator of gastric mucosal barrier stability. In this respect, it had been reported that chronic nicotine treatment led to depletion of gastric mucus (Kaunitz et al. 1993) and a reduction in mucosal thickness (Jarvis & Whitehead, 1980). In cultured rat antral mucosa, 50% inhibition of gastric mucus synthesis was observed when tobacco smoke condensate was added into the culture medium (Yeomans, 1988). Wong and colleagues (2002) found that 10 days of nicotine treatment decreased the intramucosal mucus layer in a dose-dependent manner in stressed rats, suggesting that nicotine in cigarette smoke

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could decrease the synthesis of mucus in the mucosa and adversely affect the defensive mechanism of the stomach. In contrast, other researchers (Endoh & Leung 1994; Eastwood, 1997) reported that both acute and chronic administration of nicotine was found to inhibit PGE2 production and increase susceptibility to ulceration in the stomach and perhaps increase the production of free radicals and the release of vasopressin, a potent vasoconstrictor. Reduced PGE2 was again documented in this study. Other studies revealed that cigarette smoke and nicotine reduced the levels of epidermal growth factor and prostaglandins, which are the factors essential for repair of gastric ulcers (Maity et al. 2003). Furthermore, nicotine caused a reduction in gastric mucosal blood flow and mucus volume (Thomas et al. 2005). All these factors may partly explain why cigarette smoke and its components exacerbated gastric ulceration (Chu et al. 2013). In the present study, gastric erosion, transformation of zymogen granules in peptic cells into the mucous variety, a significant increase in parietal cell numbers and gastrin expression, small haemorrhagic lesions and inflammatory cell infiltrates were most obvious in rats that received oral or I.P. nicotine administration. The more changes in the gastric mucosa seen with the oral route of nicotine administration could be attributed to the direct effect of nicotine on gastric mucosa and absorbtion of nicotine from the gastrointestinal tract when administered by the oral route. More work must be done to clarify the present variation in response, including pharmacokinetics and byproducts of nicotine upon intake by different routes. In conclusion, this study provides evidence that chronic nicotine administration by different routes can lead to a subset of clinical gastric diseases, because it increased the secretion of gastrin and ghrelin and decreased the secretion of prostaglandin E2 , which resulted in significant morphological changes in the gastric mucosa, including mucosal erosions, hyperaemia and mobilization of gastric mucosal contents of gastrin, ghrelin and histamine into the gastric lumen. Blocking gastrin receptors or inhibition of gastrin secretion may provide a new target for prevention of peptic ulcer upon exposure to nicotine in tobacco products. The role of ghrelin in peptic ulcers induced by nicotine may be protective or carcinogenic and needs further molecular investigations. References Abd El-Haleem MR, Soliman HM & Abd El Motteleb DM (2013). Effect of experimentally induced portal hypertension on the fundic mucosa of adult male albino rats and the possible protective role of quercetin supplementation: histological, immunohistochemical, and biochemical study. Egypt J Histol 36, 60–77.

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Additional information Competing interests

None declared. Author contributions

S.S.A. conducted the study, analysed and drafted the histological part of the study. E.A.H. designed the study, revised the manuscript for important intellectual content and acted as guarantor of the study, who provided input regarding the design, revised the manuscript and helped in data collection. N.N.A. helped in drafting and revision of the manuscript, helped in data collection and analysed the data. A.S. and M.I.S. helped to conduct the study. The final manuscript was approved by all authors. Funding

This study was funded by the Deanship of Scientific Research (DRS), King Abdulaziz University, Jeddah (grant number 13-140-D1432).

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Acknowledgements The authors acknowledge technical and financial support from the Deanship of Scientific Research. The authors would like to thank the following: Suzan Kamel ElSayed, Associate Professor at the Biomedical Sciences Department, Oakland

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University, William Beaumont School of Medicine for editing the manuscript; Professor Jaudah Al-Maghrabi and Miss Tedora Pedraba, Pathology Department, King Abdulaziz University and Miss Hana Khalaf, the technician in the preclinical research unit at the King Fahd Center for Medical Research (KFMRC), for providing help in immunohistochemical staining.