Food and Chemical Toxicology 58 (2013) 522–529

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Food and Chemical Toxicology

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Antioxidant effect of zinc chloride against ethanol-inducedgastrointestinal lesions in rats

0278-6915/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2013.05.022

⇑ Corresponding authors. Address: Departamento de Química, Centro de CiênciasNaturais e Exatas, Universidade Federal de Santa Maria – UFSM, 97105-900 SantaMaria, RS, Brazil. Tel.: +55 3220 8799; fax: +55 3220 8978.

E-mail addresses: rafaelineu@gmail.com (R.P. Ineu), pereirame@yahoo.com.br(M.E. Pereira).

Rafael Porto Ineu a,⇑, Cláudia Sirlene Oliveira a, Vitor Antunes Oliveira a, Lucélia Moraes-Silva a,Sônia Cristina Almeida da Luz a,c, Maria Ester Pereira a,b,⇑a Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Universidade Federal de Santa Maria – UFSM, 97105-900 Santa Maria, RS, Brazilb Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria – UFSM, 97105-900 Santa Maria, RS, Brazilc Departamento de Patologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil

a r t i c l e i n f o

Article history:Received 22 January 2013Accepted 2 May 2013Available online 29 May 2013

Keywords:StomachIntestineHistopathologyOxidative stressZinc content

a b s t r a c t

The aim of the present study was to evaluate the possible effects of zinc chloride against the gastrointes-tinal lesions caused by oral administration of ethanol in rats. Rats were divided into five groups, namely,saline, ethanol, zn, zn + ethanol and ethanol + zn. Ethanol 70% (2 mL/kg) was administered by gavage in36 h fasted rats. Zinc chloride (27 mg/kg, �13 mg/kg of zinc) was given by gavage 1 h before or 1 h afterthe administration of ethanol. Oral administration of ethanol consistently induced damage in the ratglandular stomach and intestine. Zinc did not demonstrate effect per se and significantly reduced gastro-intestinal lesions when administered either before or after lesion induction. Ethanol induced enhance-ment of thiobarbituric acid reactive substance and reactive species levels, diminished the ascorbic acidand total protein SH content as well as superoxide dismutase and catalase activity in stomach and intes-tine of rats. Zinc treatment prevented and reversed these alterations induced by ethanol. Stomach andintestine of rats treated with zinc presented higher zinc content than the tissues of rats treated only withethanol. Non-protein SH content was not altered by any treatment. Results suggested that the gastroin-testinal protective effect of zinc in this experimental model could be due to its antioxidant effect.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Gastric mucosal erosion was reported to be associated with theimbalance between the aggressive factors in the lumen and protec-tive mechanisms in the duodenal mucosa. Gastrointestinal prob-lems have now become a global problem, and many studies werecarried out towards fixing it (Alvarez-Suarez et al., 2011; Ineuet al., 2008).

The etiology of gastric damage caused by ethanol is still notfully understood; however, it has been shown to produce gastricdamage by impairing gastric defensive factors such as mucus andmucosa circulation (Cemek et al., 2010; Savegnago et al., 2006;Szabo et al., 1992). In addition, the gastric damage caused by eth-anol may be due to the increase of oxygen radicals and lipid perox-idation (Alvarez-Suarez et al., 2011; Mutoh et al., 1990). Reactiveoxygen species (ROS) provoke severe changes at the cellular levelleading to cell death because of their extreme reactivity. They at-tack essential cell constituents, leading to the formation of toxic

compounds (Kahraman et al., 2003). Both superoxide anion radicalðO��Þ2 and hydroxyl radical (�OH) are involved in tissue damagethrough initiation of lipid peroxidation and disruption of the inter-stitial matrix (Gu et al., 2012; Hogg et al., 1992). Indeed, includingour previous study and others, wide types of compounds have beentested to avoid the effects of ROS into the cells (Ineu et al., 2008;Nogueira et al., 2004; Savegnago et al., 2006).

Zinc homeostasis is important for the integrity of gastric muco-sal cells and is a key factor for the preservation of the structuralintegrity of the intestinal barrier (Lambert et al., 2004). A reductionin zinc content of the mucosa is observed in patients affected byulcerative colitis, which is associated with an increase in reactiveoxygen intermediates (Faa et al., 2008). Several key enzymes pres-ent in the epithelial cells of the intestinal mucosa, such as carbonicanhydrase are metalloenzymes and require zinc for their action(Kiefer and Fierke, 1994). Also, zinc is a cofactor for enzymes likesuperoxide dismutase, collagenase, alcohol dehydrogenase, alka-line phosphate and in spermatogenesis process (Mei et al., 2009).Zinc complexes have also been shown to have antiulcer activity.Zinc–carnosine is an antiulcer drug commonly used in the treat-ment of gastric ulcers in Japan (Odashima et al., 2006). Thezinc–indomethacin and zinc–naproxen complexes reduce moresignificantly the ulcerogenic effects compared with similar drugs,without affecting its therapeutic action (Sharma et al., 2003; Singla

R.P. Ineu et al. / Food and Chemical Toxicology 58 (2013) 522–529 523

and Wadhwa, 1995). Studies have revealed that a significant frac-tion of ingested alcohol is oxidized in gastric mucosa cells by azinc-dependent enzyme, alcohol dehydrogenase (Mutoh et al.,1990). This metabolic step is known as the first-pass metabolismof ethanol and is considered a gastrointestinal barrier against thesystemic toxicity of ethanol (Gu et al., 2012; Julkunen et al., 1985).

It has been known that an excessive intracellular increase ofzinc can be cytotoxic and cause neuronal degeneration (Choi andKoh, 1998). However, recent studies have shown that zinc can beused as an important treatment against several toxicants, such astoxic metals. In line with this, previous studies from our groupdemonstrated that zinc acts as a protector agent against severalmercury toxic effects in rats (Franciscato et al., 2011; Peixotoet al., 2003).

The aim of the present study was to evaluate the gastrointesti-nal protective effects of zinc chloride (ZnCl2) against the lesionscaused by oral administration of ethanol in rats and the possibleantioxidant effects of ZnCl2 against damage induced by ethanolin stomach and intestine of rats. In line with the previous resultsabout zinc, this is the first time that the gastrointestinal benefitseffects of ZnCl2 against lesions caused by ethanol in rats arechecked.

2. Materials and methods

2.1. Animals

A total of 34 male adult Wistar rats (225 ± 10 g) from our own breeding colonywere used (6–7 animals per group). Each cage contained five animals, on a 12-hlight: 12-h dark cycle, at a room temperature of 22–24 �C, and with free access tofood and water. The animals were used according to the guidelines of the Commit-tee on Care and Use of Experimental Animal Resources, the Federal University ofSanta Maria RS, Brazil.

2.2. Materials

Reagents used were obtained from Sigma Chemical Co. (St. Louis, MO) and stan-dard commercial suppliers. The commercial kit for biochemical dosages was ob-tained from Labtest Diagnostica SA, Brazil.

2.3. In vivo and ex vivo experiments

2.3.1. Ethanol-induced gastric lesions and zinc treatmentThe gastric lesions were induced with ethanol according to the method de-

scribed by Robert (1979). The lesions were induced in 36 h fasted rats. For this pur-pose the rats were kept in a cage equipped with a gate apparatus that hindered theanimals feeding, allowing only water. Absolute ethanol (etOH) was diluted at 70% indistilled water and administrated by gavage (v/v, 2 mL/kg, p.o.). The ZnCl2 at dose of27 mg/kg (1 mL/kg) (�13 mg/kg of Zn2+) (Peixoto et al., 2003) was dissolved in sal-ine 150 mM and administered (p.o.) 1 h before or 1 h after etOH administration. Theanimal groups were following:

Group 1� salineþ saline

Group 2� etOHþ saline

Group 3� zincþ saline

Group 4� zincþ etOH

Group 5� etOHþ zinc

In the zinc pre-treatment protocol, 1 h after the administration of ethanol, ratswere sacrificed by decapitation. In the zinc post-treatment protocol, animals weresacrificed also by decapitation 1 h after Zn administration. The tissues were imme-diately removed to measure the biochemical parameters and stomachs were usedto determine the gastric lesion index.

2.3.2. The gastric lesion indexThe stomachs were removed, opened along the greater curvature and fixed to

determine the gastric lesion index. The ulcerative lesion index of each animalwas calculated according to Gamberini et al. (1991) and using score as follows: lossof mucosal folding, mucosal discoloration, edema or hemorrhage (score 1 each); ul-cers/cm2 less than 1 mm (score = number of ulcer � 2); ulcers more than 1 mm/cm2

(score = number � 3); perforated ulcers (score = number � 4). The observer of gas-tric lesions was blinded to the treatment. The stomachs of the rats from all fivegroups were removed and examined morphologically and histological. The gastrictissue was fixed in 10% neutral formalin and embedded in paraffin. Sections fromtissue blocks were stained with hematoxylin–eosin (HE).

2.3.3. Tissue preparationAfter 1 h of the last exposure, rats were euthanized by decapitation for blood

and tissues collection. Serum was obtained by centrifugation at 2000g for 10 min(hemolyzed serum was discarded). Stomach, kidney and intestine (three portionsof beginning, medium and end of intestine) were quickly removed, cleaned andhomogenized in 10 volumes of 50 mM Tris–HCl, pH 7.4. The homogenate was cen-trifuged at 2000g at 4 �C for 10 min and a low-speed supernatant fraction (S1) wasused for ex vivo assays.

2.3.4. Renal and hepatic metabolic parametersSerum enzymes AST (aspartate aminotransferase) and ALT (alanine amino

transferase) were used as the biochemical markers for the early acute hepatic dam-age and determined by the colorimetric method of (Reitman and Frankel, 1957)).Renal function was analyzed by determining serum urea (Mackay and Mackay,1927) and creatinine levels (Jaffe, 1986) (LABTEST, Diagnostic SA, Minas Gerais, Bra-zil) by colorimetric method.

2.3.5. Zinc content determinationZinc levels were determined by inductively coupled plasma atomic emission

spectrometry (ICPE-9000; Shimadzu Scientific Instruments). The samples of wet tis-sue (0.5 g of stomach and intestine) were placed in vials and frozen at �20 �C untilanalysis. The digestion of samples and the determination of Zn content were con-ducted as described in detail by Prohaska et al. (2000) with some modifications.Samples were digested with concentrated HNO3 in an overnight water bath(100 �C). After digestion, samples were diluted with deionized water to 25 mLand transferred to graduated poly-propylene vials and determined by ICPE-9000.The analytical standard zinc (Merck�) was used to make the curve and the resultswere expressed as (lg/g of tissue).

2.3.6. Lipid peroxidationThe low supernatant fraction (S1) of stomach, intestine and kidney were used

for thiobarbituric acid-reactive species (TBARS) assay according to Ohkawa et al.(1979). Samples were incubated with 500 lL thiobarbituric acid (0.8%), 200 lLSDS (8.1%) and 500 lL acetic acid for 2 h at 95 �C. The amount of TBARS producedwas measured at 532 nm (Spectrophotometer U-2001 Hitachi), using MDA as anexternal standard.

2.3.7. Reactive species measurementFormation of reactive species (RS) was estimated according to a previous report

by Ali et al. (1992) and adapted for stomach, intestine and kidney tissues. An aliquotof S1 was incubated with 10 lL of 20 ,70-dichlorofluorescein diacetate (DCFH-DA;10 lM). The RS levels were determined by a spectrofluorimetric method. The oxida-tion of DCFH-DA to fluorescent dichlorofluorescein (DCF) was measured for thedetection of intracellular RS. The DCF fluorescence intensity emission was recordedat 525 and 488 nm of excitation 60 min after the addition of DCFH-DA to themedium.

2.3.8. Ascorbic acid determinationAscorbic acid determination was performed as previously described by Carr

et al. (1983) and Jacques-Silva et al. (2001) with some modifications. S1 proteincontent from stomach, intestine and kidney were precipitated in 10 volumes of cold4% trichloroacetic acid solution and centrifuged (S2). The medium (final volume of1 mL) was incubated for 3 h at 38 �C, after this was added 1 mL of H2SO4 65% (v/v).The reaction product was determined using a color reagent containing 4.5 mg/mLdinitrophenyl hydrazine and 0.075 mg/mL CuSO4 at 520 nm and calculated usinga standard curve (1.5–4.5 lmol/L ascorbic acid freshly prepared in sulfuric acid)(Spectrophotometer U-2001 Hitachi – Japan).

2.3.9. Thiol groupsThiol group from stomach, intestine and kidney were determined as previously

described by Ellman (1959). To determination of the total thiol groups the S1 wasused, and to determine non-protein thiol (NPSH) the protein fraction of S1 was pre-cipitated with 200 lL of 10% trichloroacetic acid followed by centrifugation. Thecolorimetric assay was carried out in 1 M phosphate buffer, pH 7.4. A standardcurve using glutathione was constructed in order to calculate the SH in the tissuesamples (Spectrophotometer U-2001 Hitachi – Japan).

2.3.10. SOD activitySuperoxide dismutase activity was assayed spectrophotometrically as previ-

ously described by Misra and Fridovich (1973). This method is based on the capac-ity of SOD to inhibit the autoxidation of epinephrine to adrenochrome. Thesupernatant (S1) (stomach, intestine and kidney) were assayed and the color

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reaction was measured at 480 nm (Spectrophotometer U-2001 Hitachi – Japan).One unit of enzyme was defined as the amount of enzyme required to inhibit therate of epinephrine autoxidation by 50% at 26 �C.

2.3.11. Catalase activityThe supernatant (S1) (stomach, intestine and kidney) were assayed (Spectro-

photometer U-2001 Hitachi – Japan) by the method of Aebi et al. (1995), which in-volves monitoring the disappearance of H2O2 in the presence of cell homogenate at240 nm. Samples of S1 were added to a cuvette and the reaction was started by theaddition of 100 lL of freshly prepared 300 mM H2O2 in phosphate buffer (50 mM,pH 7.0; total volume of incubation: 1 ml).

2.3.12. Protein determinationProtein was measured by the method of Bradford (1976) using bovine serum

albumin as the standard protein source.

2.4. Statistical analysis

Results were analyzed by the non-parametric method of the Kruskal–Wallis testwith Dunn’s post-hoc test when appropriate or were analyzed by one-way ANOVA,followed by Tukey’s post-hoc test when appropriate. Differences between groupswere considered to be significant at least when p < 0.05. Data were expressed asmedian (interquartile intervals) (non-parametric data) or as mean ± SEM (paramet-ric data).

Fig. 1. Gross appearances of stomach from groups: (A) saline,

3. Results

3.1. Effect of zinc on ethanol-induced gastric lesions

Macroscopic appearances and microscopic analysis of gastrictissue are shown in Figs. 1 and 2, respectively. Rats treated onlywith saline (Fig. 1A) or zinc plus saline (Fig. 1C) showed normalglandular stomach. Fig. 2B shows that the oral administration ofethanol 70% consistently induced damage in this structure charac-terized by longitudinal linear lesion into the gastric body and bot-tom with some haemorragic regions. Both pre- and post-treatmentof rats with ZnCl2 reduced gastric lesions induced by ethanol(Fig. 1D and E); however zinc pre-exposure was more effective.These results are shown in Table 1 as ulcer index. One-way ANOVArevealed a significant (p < 0.05) zinc protection (zn + etOH) (89%)and reversion (etOH + zn) (79%) of gastric lesions induced by etha-nol. Microscopic analysis also revealed no alterations in gastric tis-sues from saline (Fig. 2A) and zinc (Fig. 2C) treated rats.Microscopic analysis of injured areas induced by ethanol (Fig. 2B)demonstrated disruption and exfoliation of the superficial gastricepithelium. Rats pre-treated with zinc (zn + etOH) (Fig. 2D)

(B) ethanol, (C) zinc, (D) zinc + ethanol, (E) ethanol + zinc.

A B

C D

E

#

#

Fig. 2. Morphological examination of gastric mucosa of rats. Saline (A), zinc (C) and zinc plus ethanol (zn + etoh) (D) rats presented normal histological appearance of thegastric mucosa. Ethanol-treated rats (B) presented disruption and exfoliation of the superficial gastric epithelium (⁄⁄), hyperemia ( ) and moderate hemorrhage (⁄) in themucosa. Gastric mucosa of rats receiving ethanol plus zinc (etoh + zn) (E) showed no morphologic changes, only the capillaries appeared dilated (#). Details in 20�.(Hematoxylin & Eosine 10�).

Table 1Quantification of gastric lesions, serum biochemical parameters and zinc content of rats submitted to ethanol-induced gastric lesions and treated with zinc.

Groups Ulcer index AST (U/mL) ALT (U/mL) Urea (mg/dL) Creatinine (mg/dL) Zinc content (lg/g of tissue)

Stomach Intestine

Saline 0.0a 81 ± 5.1 18 ± 3.4 34 ± 1.7 0.81 ± 0.10 38.3 (35.4/66.5) 97.2 (88.3/182.5)etOH 14.0 ± 0.4b 84 ± 4.8 21 ± 4.9 35 ± 3.2 0.79 ± 0.09 25.6 (23.2/31.8) 62.9 (37.5/64.8)Zn 0.5 ± 0.2a 80 ± 6.1 16 ± 1.5 36 ± 5.5 0.89 ± 0.08 63.2 (56.5/63.7) 88.6 (49.5/565.2)Zn + etOH 1.5 ± 0.2a 86 ± 1.5 28 ± 5.2 37 ± 1.4 0.68 ± 0.12 70.1 (41.8/133.8) 141.3 (76.4/159.2)etOH + Zn 3.0 ± 0.3c 83 ± 2.1 23 ± 3.2 39 ± 2.3 0.78 ± 0.13 91.3 (69.2/111.6)� 106.4 (99.0/129.1)

Ulcer index, AST, ALT, urea and creatinine. Parametric data are reported as mean ± SEM. One-Way/ANOVA with Tukeys post-hoc test; different letters represents significantstatistical difference among groups (b and c: p < 0.05).Zinc content. Non-parametric data are reported as median (interquartile intervals). Kruskal-Wallis following by Dunn’s post-hoc test; asterisk represents significant statisticaldifference from etOH group (p < 0.05).

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showed mucosal and superficial epithelium integrity and the intactmicrocirculatory system. Rats post-treated with zinc (etOH + zn)showed no evidence of mucosa damages, whereas the capillariesappeared dilated (Fig. 2E).

3.2. Renal and hepatic metabolic parameters and zinc content

Table 1 shows that renal (urea and creatinine) and hepatic (ASTand ALT) markers parameters were not significantly altered by eth-

Fig. 3. Lipid peroxidation (TBARS) and ROS formation of stomach (A and D), intestine (B and E) and kidney (C and F) from rats exposed to induction of lesions by etOH and pre-or post-treated with zinc. Data are reported as means ± SEM. One-way/ANOVA Tukeys test; different letters represent significant statistical difference among groups (Fig. 3 Aand B b: p < 0.001; Fig. 3D and E b: p < 0.0001).

Fig. 4. Ascorbic acid and total SH protein content of stomach (A and D), intestine (B and E) and kidney (C and F) from rats exposed to induction of lesions by etOH and pre- orpost-treated with zinc. Data are reported as means ± SEM. One-way/ANOVA Tukey’s test; different letters represent significant statistical difference among groups (Fig. 4 A, B,D and E b: p < 0.001).

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anol and/or ZnCl2 treatment. Stomach and intestine zinc contentare also shown in Table 1. For stomach, Kruskal–Wallis testshowed significant effect (Dunn’s post-hoc test: p < 0.05) of treat-ment on zinc content [H(4) = 10.43, p < 0.05]. In general the groupstreated with zinc showed a slight increase of zinc content: how-ever, the significant effect only was only verified in stomach of ratspost-treated to zinc. Pre-exposure to zinc demonstrated a tendency

to enhance zinc content. However, for intestine, no difference inzinc content was observed in any groups.

3.3. Lipid peroxidation and ROS formation

Fig. 3 shows the TBARS content and ROS formation in stomach(A), intestine (B) and kidney (C) from rats exposed to ethanol and

Fig. 5. SOD and CAT activities of stomach (A and D), intestine (B and E) and kidney (C and F) from rats exposed to induction of lesions by etOH and pre- or post-treated withzinc. Data are reported as means ± SEM. One-Way/ANOVA Tukeys test; different letters represent significant statistical difference among groups (Fig. 5 A and D b: p < 0.01; Eb: p < 0.001 and F b: p < 0.05).

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pre- or post-treated with zinc. One-way ANOVA revealed thatethanol caused a significant increase in TBARS production in stom-ach [F(4,29) = 19.26 (p < 0.001)] and intestine [F(4,29) = 23.47(p < 0.001)], but not in kidney. Zinc pre-exposure preventedsignificantly this effect in both tissues and post-treatment withzinc was completely efficient only in stomach (Fig. 3A), showinga partial effect in intestine (Fig. 3B). Still, in Fig. 3 it can be observedthat the treatment with zinc was efficient to decrease the produc-tion of spontaneously ROS formation in stomach (Fig. 3D)[F(4,29) = 155.05 p < 0.0001] and intestine (Fig. 3E) [F(4,29) =81.05 p < 0.0001] by DCFH-DA addiction. Interestingly, pre-treat-ment with zinc showed a complete blockage against ethanol induc-tion. On the other hand, zinc was partially efficient in reverting theeffects of ethanol in stomach and intestine.

3.4. Ascorbic acid and Thiol content

One-way ANOVA revealed that ethanol significantly reducedthe stomach [F(4,29) = 26.01 p < 0.001] and intestine[F(4,29) = 43.75 p < 0.001] ascorbic acid levels (Fig. 4A and B). Here,only the pre-treatment with zinc was significantly able to protectthis alteration. Regarding SH content, one-way ANOVA demon-strated that ethanol caused a significant decrease in the total SHcontent in stomach [F(4,29) = 34.69 p < 0.001] and intestine[F(4,29) = 24,74 p < 0.001] (Fig. 4D and E), whereas the pre- andpost-treatment with zinc induced an increased of SH content instomach and intestine, respectively (Fig. 4D and E). No statisticaldifference was observed in NPSH for any groups in the three tissuestested (data not shown). Neither ascorbic acid (Fig. 4C) nor SH con-tents (Fig. 4F) were not changed in kidney by ethanol and zinctreatments.

3.5. SOD and catalase activity

Fig. 5A shows that SOD activity from stomach, but not fromintestine and kidney (Fig. 5B and C), presented significantly dimin-ished in ethanol group [F(4,15) = 7.928 p < 0.01]. Still, only the pre-treatment with zinc was significantly able to protect this alter-

ation. Fig. 5 also shows that CAT activity was significantly inhibitedby ethanol treatment in stomach [F(4,29) = 8.78 p < 0.01], intestine[F(4,29) = 14.35 p < 0.001] and kidney [F(4,29) = 3.94 p < 0.05](Fig. 5D–F). Zinc treatments protected this enzyme from ethanoleffects in the three tissues studies, and induced an increase of thisactivity in intestine when the animals were pre-treated.

4. Discussion

Various factors, such as the impairment of the balance betweenaggressive (increased acid secretions) and protective factors, stress,trauma, sepsis, hemorrhagic shock, burns, pulmonary and liver dis-eases, Helicobacter pylori, use of cigarettes and alcohol as well assteroidal and non-steroidal drugs have been shown to play a rolein gastric ulcerogenesis (Hooderwerf and Pasricha, 2006). Thisstudy clearly shows the ability of zinc chloride to reduce etha-nol-induced gastrointestinal lesions in rats. The mechanism of gas-trointestinal protective effect of zinc is far from clear. However,Tran et al. (2005) already demonstrated that short-term zinc sup-plementation attenuates Helicobacter felis-induced gastritis inmice.

Firstly we assessed histological status of the mucosa in responseto treatments and observed that not only macroscopical ulcer scorewas significantly decreased when zinc was administered prior toethanol, but also the microscopic picture of the gastric mucosashowed improvement of vascularisation. The macroscopic and his-topathology results show marked gastric mucosal damage in rats1 h after oral ethanol administration (Figs. 1 and 2). This led toelongated hemorrhagic lesions, confined to the glandular portionwith highest subjective ulcer-scoring. Both pre- and post-treat-ment with zinc showed impressive mucosal healing demonstratingthat pre-being more potent than post-treatment (Table 1; Figs. 1and 2). Re-epithelialization is crucial in the recovery of the gastricmucosa upon ulceration. Ulcer healing is a complex and tightlyregulated process of filling the mucosal defect with proliferatingand migrating epithelial and connective tissue cells. Growth fac-tors, such as epidermal growth factor, basic fibroblast growth fac-tor, platelet derived growth factor, and other cytokines produced

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locally by regenerating cells, control re-epithelialization and thereconstruction of glandular structures (Tarnawski et al., 2001).

It has been known that high dose of some metals (mercury,lead, cadmium, aluminum, arsenic) are toxic to various tissues,otherwise, in this study neither renal (urea and creatinine) nor he-patic (AST and ALT) markers parameters were altered by treat-ments (Table 1), indicating that kidney and liver are not targetorgans in this model of intoxication with this model of induction.This model of intoxication is very important because in an idealmodel, only gastric system should be altered. The main proposalof this study was to verify the zinc effect in gastrointestinal param-eters, indeed concomitantly to this, it is already known that oralzinc renal toxicity in rats is around 550 mg/kg (Maita et al.,1981). Thus, we verified that zinc had no toxic renal effects in thisdose tested (13 mg/kg).

Amongst various factors, oxidative stress has been implicatedfor the induction and pathogenesis of the ethanol-mediated gastro-duodenal injury (Mutoh et al., 1990; Szabo et al., 1992). Extensiveresearch has proved that antioxidants might be effective not onlyin protecting against gastric mucosal injury, but also in inhibitingthe progression of gastric ulcer (Ineu et al., 2008). Our results showan increase of TBARS and ROS along with a decrease of total SHcontent, a depletion of ascorbic acid content and an inhibition ofSOD and CAT activities in the gastrointestinal tissues after the oralethanol administration. Previous study from our group (Ineu et al.,2008) demonstrated that the increase of stomach TBARS produc-tion induced by ethanol is prevented by treatment with an organo-selenium derivative compound. In line with this, the present studyshows that the pre- and post-treatment with zinc chloride wasable to prevent and restore the TBARS and ROS formation in stom-ach and also in intestine of rats with gastrointestinal lesions in-duced by ethanol (Fig. 3).

Some authors consider that ascorbic acid is a marker of oxida-tive stress and its reduced levels are in accordance with enhancedoxidative stress (de Bem et al., 2006). In this study we verified thatstomach and intestine ascorbic acid content were reduced by oralethanol administration and only the pre-treatment with zinc wasable to normalize to control levels (Fig. 4). These results are inaccordance with our previous study (Ineu et al., 2008) that demon-strated that the diphenyl diselenide also enhanced the stomachascorbic acid content in comparison with ethanol groups. Althoughthe mechanism involved in the zinc prevention on the ethanol-in-duced decrease in ascorbic acid in stomach and intestine tissues isunknown, it is possible to consider that this effect is related to theantioxidant activity of zinc.

The induced lipid peroxidation may cause increased glutathioneconsumption. The sulfhydryl compounds help in recycling endoge-nous antioxidant vitamins, thereby, preventing lipid peroxidation.More importantly, they also protect mucus by preventing ruptureof the disulfide bridges that join the mucus subunits and maintainits structural integrity. The decrease in endogenous thiol in etha-nol-induced gastric injury and its role in mucosal protection havebeen demonstrated earlier (Al-Howiriny et al., 2010; Al Moutaeryet al., 2012; Bertrand et al., 1999; Hernandez-Munoz et al.,2000). Both pre and post-treatment with zinc provided nearly sim-ilar and significant suppression of the oxidative damages to thebiomacromolecules (Fig. 4).

The exact mechanism of gastric mucosa pathogenesis caused byethanol is not well understood. Recent studies have demonstratedthat active oxygen species may be involved in gastric mucosaldamage. To confirm the role of free radicals in the process of ulcer-ation, various experiments have been conducted using enzymaticantioxidants such as SOD and CAT (Alvarez-Suarez et al., 2011;Amaral et al., 2013; Ineu et al., 2008). In this study, we can inferthat ethanol decreased SOD activity only in stomach however,the pre-treatment with zinc could prevent this effect. Our results

also showed that the oral administration of ethanol 70% caused asignificant inhibition of gastrointestinal and renal CAT activityand the pre and post-treatment with zinc was capable of avoidingthe damage effect (Fig. 5). It has been known that zinc is necessaryto an optimum SOD activity since this enzyme is zinc dependent.Hence, an enhance of the stomach SOD activity could be explainedby an increase in the zinc levels (Table 1), whereas no increase wasobserved in the intestine, since this tissue showed to absorb morezinc than the stomach (Yasuno et al., 2011). Our results are inaccordance with Alvarez-Suarez et al. (2011) that demonstrated adecrease in stomach SOD activity caused by oral administrationof ethanol. This might decrease the ulcer progression and promotehealing of gastric lesions induced by acute intake of ethanol.

5. Conclusions

In conclusion we can infer that zinc decreases the gastrointesti-nal TBARS and ROS production due to its ability to restore theascorbic acid content and total thiol groups, leading to a restora-tion of stomach and intestine SOD and CAT activities of rats inhib-ited by oral administration of ethanol. Based on these resultstogether, we conclude that zinc was able to prevent the damage in-duced by ethanol upon the rat gastric mucosa in vivo.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Financial support-provided by CNPq, CAPES and FAPERGS isgratefully acknowledged RPI and MEP are the recipient of CNPq fel-lowship (503867/2011-0), and CSO, VAO and LM-S are the recipi-ents of CAPES fellowships.

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