Role of neutrophils in acrylonitrile-induced gastric mucosal damage

Toxicology Letters 208 (2012) 108–114

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Role of neutrophils in acrylonitrile-induced gastric mucosal damage Nadia M. Hamdy a , Fahad A. Al-Abbasi b , Hassan A. Alghamdi c , Mai F. Tolba d , Ahmed Esmat d , Ashraf B. Abdel-Naim d,∗ a

Department of Biochemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Poison Control Center, Jeddah, Saudi Arabia d Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt b c

a r t i c l e

i n f o

Article history: Received 24 September 2011 Received in revised form 23 October 2011 Accepted 24 October 2011 Available online 28 October 2011 Keywords: Acrylonitrile Gastric mucosal damage Neutrophils

a b s t r a c t Acrylonitrile (ACN) is a widely used intermediate in the manufacture of plastics, acrylic fibers, synthetic rubbers and resins that are used in a variety of products including food containers and medical devices. ACN is a possible human carcinogen and a documented animal carcinogen, with the stomach being an important target of its toxicity. ACN has been previously reported to require metabolic activation to reactive intermediates and finally to cyanide (CN− ). The current study aimed at exploring the potential role of neutrophils in ACN-induced gastric damage in rats. Experimental neutropenia was attained by injecting rats with methotrexate. This significantly ameliorated gastric mucosal injury induced by ACN. This is evidenced by protection against the increase in gastric ulcer index, myeloperoxidase (MPO) activity and CN− level. Also, neutropenia guarded against the decrease in prostaglandin E2 (PGE2), induction of oxidative stress and reduction of total nitrites and alleviated histopathological alterations in rat stomachs. These data indicate that neutrophil infiltration is, at least partly, involved in ACN-induced gastric damage in rats. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Acrylonitrile (ACN) is a widely used intermediate in the manufacture of plastics, acrylic fibers, synthetic rubbers and resins that are used in a variety of products including food containers and medical devices. Human exposure to ACN can occur during its production and use and through using ACN-containing products. Moreover, ACN has been detected in drinking water and cigarette smoke (IARC, 1999). There is inadequate evidence in humans for the carcinogenicity of ACN and therefore, ACN is possibly carcinogenic to humans [Group 2B] according to the classification of IARC (1999). Animal studies indicated that ACN is mutagenic (DuvergerVan Bogaert et al., 1981), carcinogenic (Ghanayem et al., 2002), immunotoxic (Hamada et al., 1998), embryotoxic (Saillenfait et al., 1993) and neurotoxic (Schaffer, 1975). Furthermore, ACN has been reported to induce hemorrhagic focal superficial gastric mucosal necrosis and gastric erosions in rats (Ghanayem et al., 1985a). Several studies attempted to determine the mechanism of ACNinduced toxicity and demonstrated its ability to induce oxidative stress through depletion of cellular antioxidant defenses (Jiang

∗ Corresponding author. Tel.: +20 119998505; fax: +20 224051107. E-mail addresses: [email protected], [email protected] (A.B. Abdel-Naim). 0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.10.018

et al., 1998; Pu et al., 2006). Additionally, it has been concluded that ACN is much less reactive compared to its major metabolite 2cyanoethylene oxide (CEO) (Guengerich et al., 1981; Pu et al., 2006). Whenever positive results were obtained in experimental toxicity studies, prior metabolic activation of ACN was necessary (Léonard et al., 1999). Studies have demonstrated that CYP 2E1 is the main enzyme in the cytochromes P450 (CYP450) family involved in the bioactivation of ACN (Wang et al., 2002). Although CYP 2E1 resides mainly in the liver, this organ is not the main target of ACN toxicity. Quantitative whole body autoradiographic studies in animals indicated that the gastrointestinal tract (GIT) is a potential target site of ACN toxicity (Jacob and Ahmed, 2003b). The levels of CYP 450 in the GIT are much lower than those present in the liver (Park et al., 1995). Other metabolic enzymatic and non-enzymatic routes were proposed to explain the extra-hepatic metabolism of ACN and other nitriles. These include reactive oxygen species (Mohamadin, 2001), xanthine oxidase (Mohamadin and Abdel-Naim, 2003), myeloperoxidase (Abdel-Naim and Mohamadin, 2004) and lactoperoxidase (Nasralla et al., 2009). Toxicity of nitriles, such as ACN, is accompanied by inflammation (Ghanayem et al., 1985b). One of the earliest cellular events in inflammation is the margination of leukocytes, primarily neutrophils. Activation of neutrophils is known to generate and release a number of tissue-damaging factors including reactive oxygen species and myeloperoxidase (MPO) enzyme (Klebanoff, 1986,

N.M. Hamdy et al. / Toxicology Letters 208 (2012) 108–114

1991). In vitro activation of nitriles to CN− by hydroxyl free radicals and MPO/H2 O2 /Cl− system has been previously shown (AbdelNaim and Mohamadin, 2004). Therefore, the current work was designed to investigate the potential role of neutrophils in ACNinduced gastric damage in rats. 2. Methods

109

was measured at 460 nm with a SHIMADZU® UV-1601 spectrophotometer at 25 ◦ C. MPO activity was quantified kinetically; change in absorbance was measured over a period of 2 min, sampled at intervals of 15 s (Bradely et al., 1982). The maximal change in absorbency per min was used to calculate the units of MPO activity based on the molar absorbency index of oxidized o-dianisidine dihydrochloride which equals 1.13 × 104 M−1 cm−1 . One unit of MPO is defined as that degrading one micromole of peroxide per minute at 25 ◦ C (McVey, 2001). Results were expressed as units of activity per mg protein. Protein content was determined according to Lowry et al. (1951).

2.1. Chemicals ACN 99% and methotrexate (MTX) was purchased from Sigma–Aldrich Chemical Company (St. Louis, MO). Potassium silver cyanide [KAg (CN)2 ] was obtained from ICN Pharmaceuticals (Plainview, NY, USA). o-Dianisidine, Ellman’s reagent [5,5-dithio-bis (2-nitrobenzoic acid); DTNB], Folin reagent, thiobarbituric acid were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). All other chemicals were of the highest grade commercially available. 2.2. Animals and experimental protocol Male Wistar rats weighing 180–200 g were obtained from the animal facility of El-Nasr Co. for Pharmaceutical and Chemical Industries (Cairo, Egypt). Handling and experimentation were conducted in accordance with the international ethical guidelines concerning the care and use of laboratory animals. The experimental protocol was approved by Ain Shams University. Animals were housed in a conditioned atmosphere with free access to food and water except at the night before the experiment. Initially a dose–response study was executed for selecting ACN dose: overnight fasted animals were assigned into 4 groups (8 each). Group 1 served as control, while groups 2–4 were given ACN orally at dose levels of 15, 30, and 45 mg/kg bw (bw = body weight) respectively. Animals were sacrificed 3 h after oral treatment and stomachs were quickly excised, opened along the greater curvature and rinsed with ice-cold saline. Then the stomachs were examined macroscopically for hemorrhagic lesions developing in the glandular mucosa. The length (mm) of each lesion was measured. Ulcer index was determined from the equation; Ulcer index = 10/X, where “X” is total mucosal area/total ulcerated area (Sathish et al., 2011). Neutrophil depletion: Rats were made neutropenic by administration of MTX (2.5 mg/kg/day; i.p.) for 3 consecutive days. Control animals were injected with 0.9% saline. In all animals, neutrophils were counted before injection of MTX and on the fifth day after the final dose of MTX. Blood was collected from the tail vein and smeared on a glass slide. The smear was stained with May–Grünwald–Giemsa stain and examined under 100× objective of a light microscope. Neutropenia was confirmed by counting the number of circulating neutrophils per 100 white blood cells for each animal. Animals were deprived of food, but not water, for 16 h prior to the experiments. After 16 h of starvation period, the rats were treated either with the vehicle (Group 1) or ACN (30 mg/kg) (Group 2) orally. Group 3 contained neutrophil-depleted animal. Group 4 contained MTX-pretreated animals given ACN. All animals were sacrificed 3 h after treatment and the stomachs were removed either for the immediate determination of lesion index, or were stored at −70 ◦ C for subsequent biochemical assays. Representative stomachs from each group were fixed in 10% formol saline for subsequent histopathological examination. 2.3. Cyanide determination CN− concentration in the gastric tissue homogenates (10% w/v in phosphate buffered saline) was determined electrochemically as described by Abreu and Ahmed (1980). Briefly, 2 mL of the homogenate was added to 2 mL 4 N H2 SO4 contained in the outer chamber of a Conway microdiffusion cell (Thomas Scientific, Philadelphia, PA, USA). The inner chamber contained 2 mL 0.1 N NaOH. The cells were sealed with a glass cover by using silicone grease and rotated at 0.8 × g for 2 h. Electrodes (Orion silver sulfide electrode model 9416 BN and Orion double junction reference electrode model 9202, purchased from Orion Research, MA, USA) were placed in the inner chamber of the Conway microdiffusion cell to which 50 ␮L KAg (CN)2 indicator solution had been added. The indicator solution contained 1.25 M Na2 HPO4 , 0.55 M NaOH, and 0.46 mM KAg(CN)2 in a final volume of 100 mL. A Hanna pH meter (model 8417) was used for millivolts determinations. The CN− content of samples was determined from a standard curve constructed by the use of NaCN. The electrode response was linear with respect to logarithm of the concentration of CN− down to 0.5 nmol/mL. 2.4. Biochemical assays 2.4.1. Gastric mucosal myeloperoxidase (MPO) The entire gastric tissue was homogenized for 10 min in ice bath (10%, w/v) in 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide with a Glas-Col® homogenizer. Tissue suspensions were centrifuged at 40 000 × g for 15 min. An aliquot of 0.1 mL of the supernatant was added to 2.9 mL of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine dihydrochloride, serving as MPO substrate, and 0.0005% H2 O2 . The change in absorbance

2.4.2. Prostaglandin E2 (PGE2) determination PGE2 were quantified in the collected homogenate using a quantitative binding PGE2 enzyme immunoassay kit (Assay Designs). The kit uses a monoclonal antibody to PGE2 to bind, in a competitive manner, the PGE2 in the sample, standard, or an alkaline phosphatase molecule which has PGE2 covalently attached to it. After a simultaneous incubation at room temperature the excess reagents were washed away and substrate was added. After a short incubation time the enzyme reaction was stopped by addition of stop solution and the yellow color generated read on a microplate reader at 405 nm. The intensity of the bound yellow color is inversely proportional to the concentration of PGE2 in either standards or samples. The measured optical density of the standards was used to calculate the concentration of PGE2 in the sample (Chard, 1990; Tijssen, 1985). 2.4.3. Assessment of gastric oxidative status 2.4.3.1. Glutathione system. i. Total glutathione: Total glutathione in the gastric tissue homogenate was determined according to the method described by Tietze (1969). Briefly, 200 ␮L gastric tissue homogenate was added to 2.5 mL 0.05 mM phosphate buffer (pH 7.5), 0.8 mL 1.0 mM Na–EDTA (pH 7), 30 ␮L of 0.1 M Ellman’s reagent and 100 ␮L GSSG reductase (2.2 units/mL); the reaction was started by the addition of 100 ␮L 5.0 mM NADPH. The rate of change in absorbance at 412 nm was monitored for 5 min and compared with a standard curve. ii. Gastric mucosal glutathione (GSH): Gastric mucosal GSH was determined as protein-free sulfhydryl content using 5,5-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent) that is reduced by the SH-group in GSH to form 5-thio-2nitrobenzoic acid, which has a stable yellow color measured colorimetrically at 412 nm as described by Ellman (1959). In detail, protein precipitation was attained by mixing equal volumes of 10% aqueous homogenate and 10% trichloroacetic acid–0.005 M EDTA solution followed by centrifugation at 600 × g for 15 min. To 0.5 mL of the resulting supernatant, 0.85 mL phosphate buffer (0.1 M, pH 8) and 0.05 mL Ellman’s reagent (10 mM) were added in a microcuvette and the optical density was measured at 412 nm. iii. Oxidized glutathione (GSSG): Oxidized glutathione (GSSG) is obtained by the simple difference [total GSH = reduced GSH + GSSG]. iv. Glutathione peroxidase (GPx): The activity of GPx was determined in cytosolic fraction of gastric mucosa prepared by mixing equal volumes of 10% aqueous homogenate and Tris–EDTA buffer, pH 7.6 (100 mM Tris and 0.2 mM EDTA) followed by centrifugation at 105,000 × g at 4 ◦ C for 15 min (Moghadasian and Godin, 1996). The resulting supernatant is the cytosolic fraction. GPx activity was determined by following the rate of oxidation of glutathione by H2 O2 using NADPH in the presence of GPx. The reaction mixture for four tests was prepared as follows: to 2.59 mL phosphate buffer, pH 7 (0.05 M KH2 PO4 /Na2 HPO4 ·2H2 O and 0.005 M EDTA), the following solutions were added in turn 0.1 mL NADPH solution (0.0084 M), one unit of GR, 0.01 mL NaN3 solution (1.125 M), and finally 0.1 mL GSH solution (0.15 M). Cytosolic fraction (15 ␮L completed to 25 ␮L with phosphate buffer, pH7) was mixed with 0.7 mL of the reaction mixture in a microquartz cuvette. Finally, the enzymatic reaction was initiated by the addition of 25 ␮L of H2 O2 (0.0022 M). The conversion of NADPH to NADP+ was followed by recording the decrease in absorbance of the system at 340 nm for 3 min (Paglia and Valentine, 1967).

2.4.3.2. Catalase (CAT). Catalase activity was determined in the cytosolic fraction of gastric mucosa by recording the decomposition of H2 O2 at 240 nm with a modified method of Aebi (1984) and Luck (1963). CAT in the 10% homogenate (0.5 mL) reacted with a known quantity of H2 O2 (0.5 mM/L diluted 1000 times before use). The reaction was stopped after exactly 1 min with CAT inhibitor. In the presence of horse radish peroxidase 0.1 mL remaining H2 O2 reacts with 0.5 mL 3,5-dichloro-2hydroxybenzene sulfonic acid (DHBS) (100 mM/L in phosphate buffer pH 7.0) and 4-aminophenazone (AAP) to form a chromophore at 37 ◦ C within 10 min with a color intensity inversely proportional to the amount of catalase in the original sample. Specific CAT activity was expressed in terms of mU/mg protein.

2.4.3.3. Superoxide dismutase (SOD). Superoxide dismutase activity was determined in the cytosolic fraction of gastric mucosa through inhibition of pyrogallol autoxidation (Marklund and Marklund, 1974). Cytosolic fraction (20 ␮L) was added to a microcuvette containing 10 ␮L pyrogallol solution (10 mM dissolved in 10 mM HCl) and 1 mL Tris–HCl buffer (50 mM, pH 8.2) containing 1 mM

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0.6

diethylenetriaminopentaacetic acid. The change in absorbance per minute at 420 nm was recorded for 2 min.

2.4.4. Determination of protein The protein content of the different fractions, resulting from ultracentrifugation of mucosal homogenate, was determined using the method of Lowry et al. with bovine serum albumin as standard (Lowry et al., 1951). 2.5. Histopathology Slices of the glandular stomach from eight animals of each group was carefully dissected free of other tissues, opened longitudinally, pinned to a piece of corkboard, were fixed in 10% buffered formalin for 24 h, washed in tap water, then serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 ◦ C in hot air oven for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns by slidge microtome. The obtained tissue sections were collected on glass slides, deparaffinized, and stained by hematoxylin and eosin (H&E) stains (Banchroft et al., 1996). 2.6. Statistical analysis All data are expressed as means ± SD. Multiple group comparisons were carried out using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test for post hoc analysis. Statistical significance was accepted at a level of P < 0.05. All statistical analyses were performed using GraphPad InStat software, version 3.05 (GraphPad Software, Inc., La Jolla, CA, USA). Graphs were sketched using GraphPad Prism software, version 5.00 (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results 3.1. Gastric ulcer index Oral administration of ACN resulted in acute focal superficial mucosal erosion in rat glandular stomach. The incidence and severity of gastric damage were dose dependent (Fig. 1). Gastric lesion was comparable at dose levels of 30 and 45 mg/kg. The lower dose (30 mg/kg; p.o.) was selected and used throughout the work. The severity of gastric lesions induced by ACN administration in MTXpretreated rats was significantly lower than that in ACN-only group by about 35% (Table 1). 3.2. Neutropenia The count of neutrophils per 100 cells was 56 ± 15.16 cells in control rats. While, on day 5 of MTX administration, neutrophils count was significantly reduced to 13.33 ± 3.26 (Table 1). MTX treatment appeared to produce significant reduction in

0.4

a 0.2

m 45

30 A

A

C N

C N

C N A

g/ kg

g/ kg m

m

g/ kg

on tr ol

0.0

C

2.4.3.5. Total nitrite/nitrate (NOx) determination. Nitric oxide was determined in 10% aqueous homogenate according to Miranda et al. (2001) using zinc sulfate (ZnSO4 ) instead of ethanol for protein precipitation. The method employs the reduction of any nitrate to nitrite by vanadium after protein separation followed by the detection of total nitrite by diazotization with sulfanilamide at acidic pH followed by coupling with N-1-(naphthyl)ethylenediamine dihydrochloride (NEDD). The formed azo derivative can be measured colorimetrically at 540 nm. In detail, 0.5 mL each of supernatant, resulting from centrifugation of 10% aqueous homogenate at 21,000 × g for 15 min was treated with 50 ␮L 30% ZnSO4 for protein precipitation. Then, precipitated protein was removed by centrifugation at 21,000 × g for 15 min. The resulting supernatant (100 ␮L for tissue) was diluted to 300 ␮L with double distilled water and treated with 300 ␮L VCl3 (0.8 g% in 1 M HCl), followed by rapid addition of 150 ␮L sulfanilamide (2% in 5% HCl) followed by 150 ␮L NEDD (0.1%). The mixtures were then incubated at 37 ◦ C for 30 min then cooled. The absorbance of the formed pink chromophore was measured at 540 nm.

Ulcer index

2.4.3.4. Gastric mucosal lipid peroxides. Lipid peroxidation was estimated spectrophotometrically using the thiobarbituric acid reactive substance (TBARS) method, as described by Uchiyama and Mihara (1978). Results expressed in terms of mmol MDA formed per mg protein gastric mucosal tissue and as nmol/L in plasma. Calibration was done using 1,1,3,3-tetraethoxypropane as a standard. Briefly, the homogenate was supplemented with 0.75 g/L TBA in 0.1 mol/L HCl. The reactants were then supplemented with 5 mL n-butanol-pyridine mixture, shaken vigorously for 1 min and centrifuged for 10 min at 1700 × g. Absorbance was then read at 532 nm.

a, b

a, b

15

110

Fig. 1. Ulcer index after administration of different doses of ACN in rats. Data are mean ± SD (n = 8). (a) Significantly different from control at P < 0.05 and (b) significantly different from ACN (15 mg/kg) at P < 0.05.

neutrophils count without any significant effects on eosinophils or macrophages. 3.3. Cyanide generation in gastric tissues Cyanide determination in gastric tissue homogenates revealed that CN− level in normal rats administered ACN was significantly higher than that of neutropenic rats given the same dose of ACN by about 47% (Table 1). 3.4. Gastric MPO activity MPO enzyme activity, an indicator of neutrophil infiltration, was significantly increased in ACN-treated rats 3-fold compared to control and 4-fold compared to MTX-treated rats. MTX-pretreatment significantly prevented ACN-induced increase in MPO activity by 44% compared to ACN-only group (Fig. 2). 3.5. Gastric PGE2 level ACN-administered rats exhibited significant reduction in gastric PGE2 levels by 95% compared to control and by 85% compared to MTX-treated rats. MTX-pretreatment significantly counteracted ACN-induced reduction in PGE2 level 5-fold compared ACN-only group (Fig. 3).

Table 1 Circulating neurophils count, gastric tissues’ cyanide generation and ulcer index after ACN (30 mg/kg) administration in normal and MTX-pretreated rats compared to control and MTX groups. Groups/treatment

Neutrophils count per 100 cells

Control MTX ACN MTX − ACN

56 13 57 13

± ± ± ±

15 3a 14 3a,c

CN− (pmol/mg protein)

Gastric ulcer index

– – 40 ± 9 21 ± 4c

0.1 0.1 0.4 0.2

Data are mean ± SD (n = 8). a Significantly different from control at P < 0.05. b Significantly different from MTX at P < 0.05. c Significantly different from ACN-alone at P < 0.05.

± ± ± ±

0.01 0.14 0.09a,b 0.05a,b,c

111

2.0

a

2.0

b

1.5 1.0

a

0.5

Tissue nitrate& nitrite (µmol/mg protein)

2.5

1.5

a, b

a, b a

1.0 0.5

C N A

M TX -A C N

C

TX M

M

ol on tr

N A C

C N A

TX M

C

TX

0.0

0.0

on tr ol

MPO activity (U/mg protein)

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Fig. 2. Gastric MPO activity after the administration of ACN in normal or MTXpretreated rats. Data are the mean ± SD (n = 8). (a) Significantly different from control at P < 0.05 and (b) significantly different from ACN-alone at P < 0.05.

with MTX then administered ACN showed less reduction in gastric SOD levels by about 62% compared to ACN only treated rats (Table 2).

3.6. Gastric oxidative status 3.6.1. Glutathione system ACN group showed significant reduction in GSH and GPx gastric levels by 66.7% and 55% respectively compared to control. Moreover, the levels in the same group were reduced by 70% and 52% compared to MTX-treated rats. However, administration of ACN to MTX-treated group was accompanied by significant decrease in GSH and GPx by 31.7% and 10.95% respectively compared to control. Gastric GSSG level was significantly increased in the ACN group 10 times as control and MTX group. However, administration of ACN to MTX-treated group was associated with smaller increase in GSSG by 1.9 times compared to ACN-alone group (Table 2). 3.6.2. CAT and SOD activity ACN group showed significant reduction in gastric CAT level by 44% and 32% compared to control and MTX-treated animals respectively. Moreover, upon the administration of ACN to neutropenic rats CAT level was significantly less reduced by 40% compared to ACN and 10% compared to MTX. In addition, ACN-treatment significantly reduced gastric SOD levels by about 69% compared to control and by 62% compared to MTX-treated rats. Rats pretreated

PGE2 (Pg/mg protein)

6

4

a, b

2

a, b a N -A C

C N M

TX

A

TX M

on t

ro

l

0

C

Fig. 4. Gastric NOx levels after ACN administration to normal or neutropenic rats. Data are the mean ± SD (n = 8). (a) Significantly different from control at P < 0.05 and (b) significantly different from ACN-alone at P < 0.05.

Fig. 3. Gastric PGE2 level after the administration of ACN in normal or MTXpretreated rats. Data are the mean ± SD (n = 8). (a) Significantly different from control at P < 0.05 and (b) significantly different from ACN-alone at P < 0.05.

3.6.3. Gastric lipid peroxidation products Gastric MDA levels, an early index of lipid peroxidation, were significantly elevated in ACN group 2.6-fold compared to control. In MTX-treated animals levels were increased 1.1-fold compared to control animals. However, treatment of animals with MTX before ACN administration compromised gastric lipid peroxidation by 57% compared to ACN-only treated group (Table 2). 3.6.4. Gastric nitrate and nitrite levels (NOx) Gastric levels of NOx were significantly decreased in ACN group about 6-fold compared to control and 1.5-fold compared to MTXtreated. MTX treatment before ACN alleviated its reduction in NOx levels by 33% compared to ACN alone (Fig. 4). 3.7. Histopathology Normal histology without any histopathological alterations was observed in glandular stomach specimens from control group (Fig. 5a). However, edema and dilated blood vessels were evidenced in the submucosa of specimens from MTX-treated rats (Fig. 5b). Examination of specimens from ACN-treated rats showed degeneration and atrophy in the glandular epithelium together with edema and diffuse inflammatory cells infiltration (Fig. 5c). Specimens from neutropenic rats administered ACN showed mild inflammatory cells infiltration and dilated blood vessel in the submucosal layer with minimal atrophy in glandular epithelium (Fig. 5d). 4. Discussion Biotransformation of chemicals to more active metabolites is known to play a pivotal role in their toxicity (Ghanayem and Hoffler, 2007). Acrylonitrile (ACN) has been shown to be activated to cyanide (CN− ) by hepatic CYP 450 (Abreu and Ahmed, 1980; Suhua et al., 2010). However, alternative metabolic pathways for the biotransformation of ACN have been proposed to explain the toxicity of ACN at extra-hepatic target tissues (Nasralla et al., 2009). MPO has been proven to activate chloroacetonitrile; a structurally related compound (Abdel-Naim and Mohamadin, 2004). The current study was designed to explore the potential role of neutrophils in ACN-induced gastric damage in rats. Neutropenia was induced using MTX and ACN-induced gastric lesions were assessed. It was

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Table 2 Effect of ACN administration to normal or neutropenic rats on gastric reduced and oxidized glutathione, glutathione peroxidase, CAT, SOD and gastric lipid peroxidation product (MDA). Groups/parameters

GSH (mmol/mg protein)

Control MTX ACN MTX + ACN

64 69 21 44

± ± ± ±

17 10b 6a,c 14a,b,c

GSSG (mmol/mg protein) 4 5 45 23

± ± ± ±

1 1b 15a,c 7a,b,c

GPx (U/mg protein) 338 321 152 300

± ± ± ±

29 26b 38a,c 68b

CAT (mU/mg protein) 10 9 6 9

± ± ± ±

3 3b 2a 3b

SOD (mU/mg protein) 1981 1620 615 1623

± ± ± ±

421 377b 257a,c 473b

MDA (mmol/mg protein) 27 30 70 30

± ± ± ±

10 10b 22a 11b

Data are the mean ± SD (n = 8). a Significantly different from control at P < 0.05. b Significantly different from ACN-alone at P < 0.05. c Significantly different from MTX at P < 0.05.

found that the severity of the gastric mucosal damage induced by ACN in MTX-pretreated rats was significantly lower than that of ACN-only group. This indicates that neutropenia has an ameliorating effect against ACN-induced gastric damage. This gains support by previous studies (Alican et al., 1995; Kvietys et al., 1990; Tepperman et al., 1993). Activation of neutrophils is known to generate and release a number of tissue-damaging factors including reactive oxygen species such as superoxide anion, hydrogen peroxide, hypochlorous acid, as well as enzymes such as MPO and proteases (Elsbach and Weiss, 1988; Harlan, 1985). Neutrophils also migrate out of the microvasculature into the surrounding tissue, resulting in further disruption of the gastric tissue (Anderson et al., 1991; Klebanoff, 1986, 1991; Wallace et al., 1990). In the present work, the role of neutrophils in ACN-induced gastric lesions was further substantiated by the determination of MPO activity and CN− generation in gastric tissues. Reduction in MPO activity was concomitant with decrease in CN− generation in gastric tissues of neutropenic rats compared to ACN-treated normal rats.

This may support our hypothesis that ACN-induced gastric damage involves neutrophils activation and increased MPO activity with subsequent biotransformation of ACN to CN− . ACN has been shown to be metabolized by cytochrome P450 2E1 (Wang et al., 2002) and lactoperoxidase (Nasralla et al., 2009). It is noteworthy to indicate that MTX does not affect the activity of these enzymes. Therefore, it is not expected to have a direct effect on ACN metabolism. It was found that MTX-pretreatment prevented ACN-induced depletion of PGE2 levels in gastric mucosal tissues. Prostaglandins have been previously shown to inhibit neutrophils activation and superoxide anions generation (Grglewski et al., 1987; Kainoh et al., 1990). This may help explain the notion that neutropenia has an ameliorating role in ACN gastric injury. ACN-induced gastric damage was coupled with a marked decrease in gastric GSH levels, GPx, CAT and SOD activity. This occurred together with a significant elevation in GSSG and MDA levels in gastric mucosal tissues. Induction of oxidative stress by ACN is in agreement with previous reports. Ghanayem et al. reported

Fig. 5. (a) Sections of rat glandular stomach from control rat showing normal histological structure of mucosa and gastric glands (m) and muscular layer (ms) (H&E 40×). (b) Sections of rat glandular stomach from MTX-treated group showing edema (o) and dilated blood vessels (v) in the submucosa (H&E 40×). (c) Sections of rat glandular stomach from ACN (30 mg/kg) treated group showing atrophied glands (a) and edema (o) inflammatory cells infiltration in submucosa (arrow) (H&E 40×). (d) Sections of glandular stomach submucosa from neutropenic rats treated with ACN (30 mg/kg) showing edema (o), inflammatory cells infiltration (arrow) and dilated blood vessels (v) in submucosa (H&E 40×).

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that ACN-induced gastric lesions were associated with a significant decrease in gastric GSH levels (Ghanayem et al., 1985a). Other study in rat primary glial cells revealed that ACN enhanced MDA accumulation, and depleted GSH (Esmat et al., 2007). ACN-induced oxidative stress was also reported in rat brain (El-Sayed el-SM et al., 2008; Jacob and Ahmed, 2003a; Pu et al., 2009). On the other hand, MTX pretreatment ameliorated the ACN-induced reduction in GSH and elevation in GSSG and MDA. Reduction of circulating neutrophil numbers by MTX also resulted in less significant reduction in GPx, CAT, and SOD activity in gastric tissues. This is consistent with the fact that neutrophils infiltration and activation is associated with the release of a wide range of tissue damaging free radicals (Elsbach and Weiss, 1988; Harlan, 1985). Putting these together can further support the role of neutrophils in ACN-induced oxidative stress and tissue damage in gastric mucosa. Nitric oxide (NO), an endogenous vasodilator with anti-adhering and anti-aggregating effects on neutrophils, has been shown to play a role in preventing gastric mucosal injury (Moncada et al., 1991). Therefore, the potential effects of ACN on nitrate/nitrite (NOx) levels in gastric tissues were investigated. Gastric levels of NOx were significantly depleted in the ACN-only group. Induction of neutropenia by MTX pretreatment alleviated reduction in gastric NOx levels by ACN. This is in line with Tepperman et al. (1993) who reported that neutropenia protected against ethanol-induced gastric lesions through maintenance of NO synthase activity. The protection afforded by neutropenia can be explained by a previous study suggesting that NO production has been shown to be inactivated by cytotoxic products of neutrophils, including superoxides (Rubanyi and Vanhoutte, 1986). Neutropenia is likely to remove a major source of superoxide within the gastric mucosa leading to maintenance of NO forming capacity. Histopathological examination of specimens from rat gastric mucosa revealed that ACN caused degeneration and atrophy in the glandular epithelium together with diffuse inflammatory cells infiltration. The effect of ACN on gastric tissue histopathology is in agreement with the results of Ghanayem et al. (1985a) who reported a focal superficial gastric mucosal necrotic lesions characterized by wedge-shaped areas of acutely necrotic gastric glands in the mucosa of ACN treated animals. Neutropenic rats administered ACN showed mild inflammatory cells infiltration with minimal atrophy in glandular epithelium. This supports the observed reduction in gastric ulcer index and MPO activity which is an indicator of neutrophil accumulation (Goulet et al., 1994). In conclusion, the current study highlights an important role for neutrophils in ACN-induced gastric damage in rats. This is evidenced by the notion that neutropenia resulted in protection against the increase in ulcer index, MPO activity and CN− generation in gastric tissues. Further, neutropenia guarded against the decline in PGE2, induction of oxidative stress and decrease of NOx and alleviated histopathological alterations in rat stomachs. Conflict of Interest None to declare. Study association This study is an independent basic regular research paper and not a part of any thesis. Acknowledgements This work was supported by the Deanship of Scientific Research, project number 120/130/1431, King Abdulaziz University, Jeddah, Saudi Arabia. The authors would like to thank Prof. Adel B.

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Kholoussy, Professor of Pathology, Cairo University for his help in the histopathological examination of the specimens.

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