Effects of acute alcohol consumption and vitamin E co-treatment on oxidative stress parameters in rats tongue

Food and Chemical Toxicology 47 (2009) 1058–1063

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Food and Chemical Toxicology

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Effects of acute alcohol consumption and vitamin E co-treatmenton oxidative stress parameters in rats tongue

V.C. Carrard a,b,*, A.S. Pires a,b, M. Mendez a,b, F. Mattos a, J.C.F. Moreira b, M. Sant’Ana Filho a

a Laboratório de Histopatologia Prof. Dr. J.J. Barbachan (Patologia Bucal), Faculdade de Odontologia, Universidade Federal do Rio Grande do Sul (UFRGS),Porto Alegre, Brazilb Centro de Estudos em Estresse Oxidativo, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 April 2008Accepted 25 January 2009

Keywords:Ethanola-TocopherolFree radicalsOral mucosaOxidative stress

0278-6915/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fct.2009.01.033

Abbreviations: CAT, catalase activity; DNPH, dinitrsignificant difference; ROS, reactive oxygen substanceactivity; TBARS, thiobarbituric acid-reactive substanc

* Corresponding author. Address: Laboratório deBarbachan (Patologia Bucal), Faculdade de OdontologiaGrande do Sul (UFRGS), 90035-003 Porto Alegre, Brazi+55 51 3308 5023.

E-mail address: [email protected] (V.C. Carra

The aim of this study was to evaluate the effects of acute alcohol consumption and vitamin E co-treat-ment upon oxidative stress parameters in rats tongue. Thirty-eight, Wistar rats were separated into fivegroups (alcohol, alcohol/vitamin E, control, Tween, vitamin E). Alcohol and alcohol vitamin E groups hadthe standard diet, and 40% alcohol on drinking water. Other groups were fed with the same standard dietand water ad libitum. Vitamin E was given by gavage to vitamin E and alcohol/vitamin E rats twice aweek. Alcohol and control groups were subjected to saline gavage and Tween group to 5% Tween 80 solu-tion, the vitamin E vehicle. At day 14, the animals were anesthetized and specimens were obtained fromtongue. Lipid peroxidation (TBARS), protein oxidative damage, catalase (CAT) and superoxide dismutase(SOD) activities were quantified. Alcohol group decreased TBARS in relation to control group and alcoholvitamin-treated animals decreased TBARS when compared to Tween and vitamin E groups. SOD activitywas lower and CAT activity was higher in animals treated with both alcohol and vitamin E. These resultssuggest that short-term alcohol consumption decreases lipid peroxidation levels. Alternatively, alcohol/vitamin E group increased CAT, showing the toxicity of this association.

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1. Introduction

Oxidative stress has been suggested as playing a central role inmany pathways of alcohol-induced damage in a variety of systems,such as liver (Dey and Cederbaum, 2006), central nervous system(Nordman, 1987; Chu et al., 2007) and testes (Nordmann et al.,1990). Alcohol metabolization generates reactive oxygen species(ROS), which may damage macromolecules in the cell, includinglipids, proteins and DNA (McDonough, 2003; Wu and Cederbaum,2003; Das and Vasudevan, 2007).

It has been demonstrated that both acute and chronic alcoholexposure increase ROS production and decrease antioxidant de-fenses leading to oxidative stress in liver (Halliwell, 1991; Wuand Cederbaum, 2003). Antioxidant enzymes exhibit reduced

ll rights reserved.

ophenylhydrazine; LSD, leasts; SOD, superoxide dismutasees.

Histopatologia Prof. Dr. J.J.. Universidade Federal do Rio

l. Tel.: +55 51 3308 5011; fax:

rd).

activity in addition to decreases in the cellular and extracellularcontent of non-enzymatic antioxidants that should be obtainedby diet but are inadequately absorbed by the gastrointestinal sys-tem due to alcohol consumption (Wu and Cederbaum, 2003).

Few studies have assessed the short-term effects of alcohol onoral mucosa and has focused only on epithelial permeability (Duet al., 2000; Howie et al., 2001). Biochemical imbalance could bethe first signal of alcohol damage.

Therefore, the aim of this study was to assess the acute alcoholintake effects and the possible protective effect of vitamin E co-administration upon oxidative stress parameters and antioxidantenzymes activities after acute alcohol exposure in rats tonguetissues.

2. Materials and methods

2.1. Chemicals

Alcohol was purchased from Cromoline-Química Fina (Diadema, SP, Brazil).Thiobarbituric acid, folin–ciocalteu reagent, 1,1,3,3-tetramethoxypropane, catalase,adrenaline, dinitrophenylhydrazine (DNPH) and vitamin E (a-tocopherol) were pur-chased from Sigma Chemical Co. (St. Louis, MO, USA). Glycine was purchased fromNuclear (Diadema, SP, Brazil). Tween 80 was purchased from Riedel-de-Haën (See-lze, Germany).

Table 2Standard chow diet and solutions (water and 40% ethanol solution) consumption.

Group n Standard chow diet animal/day(g)

Solution animal/day(ml)

Control 6 12.53 37.92

V.C. Carrard et al. / Food and Chemical Toxicology 47 (2009) 1058–1063 1059

2.2. Animals and treatment

Thirty-eight, 3 months old female Wistar rats (Rattus norveggicus), weighing190–260 g, were housed in a temperature-controlled room (24 �C ± 1 �C) with12:12 h reverse light/dark cycle. This experiment was approved by the ResearchCommittee and the Ethics Committee at the Faculdade de Odontologia, UniversidadeFederal do Rio Grande do Sul (protocol no. 190/05 of Dentistry Graduate Program).

All animals used in the experiments were treated in accordance with The Guide-lines for the Care and Use of Laboratory Animals prepared by the National Academy ofSciences and published by the National Institutes oh Health.

Animals were assigned to one of the following five groups by the stratifiedweight randomization method:

(1) Control, n = 6;(2) Alcohol, n = 10;(3) Tween 80 (vitamin E solvent), n = 6;(4) Vitamin E + Tween 80, n = 6;(5) Alcohol/vitamin E, n = 10.

The control, Tween and vitamin E groups were fed a standard laboratory chowdiet for rodents (Nuvilab/CR1, Nuvital Nutrientes LTDA, Colombo, Brazil) and tapwater ad libitum. The other experimental groups (Alcohol and alcohol/vitamin E)received the same standard diet but the water was replaced with 40% (vol./vol.)ethyl alcohol throughout experiment, which was available ad libitum as well. The40% alcohol concentration was chosen because it is the same concentration foundin ‘‘cachaça”, a distilled liquor most frequently consumed by the Brazilian popula-tion (Neves et al., 1989). During the first week, the alcohol concentration was in-creased gradually from 5% to 40%, according to the Table 1 (adapted fromMcMillen et al., 2005).

Vitamin E (a-tocopherol dissolved in 5% Tween 80 solution) was given orally viagavage (200 mg/kg, twice a week according to Kalender et al. (2004) to alcohol/vita-min E and vitamin E rats. Alcohol and control groups were subjected to saline ga-vage. Tween treated animals received 5% Tween 80 (Krishnamurthy and Bieri,1963) solution at the same dose as alcohol/vitamin E and vitamin E animals withthe aim to evaluate the effect of the vitamin E vehicle.

The solutions volume (ml) and standard chow diet amount (g) consumptionwere monitored along study. Animals weight (g) was measured at the beginningand at the end of the experiment, as well as the weight gain (%) during to studywas measured.

At day 14, under intra-peritoneal anesthesia using 100 mg/kg ketamine/50 mg/kg xylasine, a specimen was obtained from tongue dorsum by 5-mm biopsy punch,which includes epithelial and connective tissues. This anatomic site was choosen inorder to standardize the location and size of the specimen. The tongue biopsy washomogenized in ice-cold phosphate saline buffer (PBS, pH 7.4), sonicated in ice bath(4 cicles of 10 s) and stored at �80 �C for further analyses. It was assessed the Lipidperoxidation (thiobarbituric acid-reactive substances-TBARS), protein oxidativedamage (carbonyl groups), catalase (CAT) and superoxide dismutase (SOD)activities.

2.3. Thiobarbituric acid-reactive substances (TBARS)

As an index of lipid peroxidation we used the formation of TBARS during anacid-heating reaction (Valenzuela, 1991). Briefly, 300 lL of sample was mixed with600 lL of trichloroacetic acid 15% and 500 lL of thiobarbituric acid 0.67%, thenheated in a boiling water bath for 20 min. TBARS were determined by absorbanceat 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. Results wereexpressed as malondialdehyde equivalents per milligram of protein.

2.4. Protein carbonyls

Oxidative damage to protein was assessed by determination of carbonyl groupsbased on their reaction with dinitrophenylhydrazine (DNPH), as described by Le-vine et al. (1990). Briefly, proteins were precipitated by the addition of 20% trichlo-roacetic acid to 150 lL of concentrated sample and reacted with DNPH. Then 8 Murea was added to samples and the carbonyl content was determined from absor-bance at 370 nm using a molar absorption coefficient of 22,000 M�1. Urea was usedinstead of guanidine hydrochloride due to higher cost of the former.

2.5. Catalase (CAT) and superoxide dismutase (SOD) activities

In order to determine CAT activity, tissue samples were sonicated in 50 mMphosphate buffer and the resulting suspension was centrifuged at 6200�g for10 min. The supernatant was used for enzyme assay. CAT activity was measured

Table 1Alcohol concentration increase per day in adaptation phase.

Day 1st 2nd 3rd 4th 5th 6th 7th

5% 10% 15% 20% 25% 30% 40%

by the rate of decrease in hydrogen peroxide (10 mM) absorbance at 240 nm (Aebi,1984). SOD activity was quantified by inhibition of adrenaline self-oxidation absor-bance at 480 nm and the tissue samples were not sonicated for this assay (Misraand Fridovich, 1972).

2.6. Protein quantification

All of the results were normalized for protein content (Lowry et al., 1951).

2.7. Statistical analysis

Analysis of variance (ANOVA) was used to compare means between groupswhen data followed normal distribution. Statistical significance was consideredwhen p < 0.05. The Tukey test was performed for multiple comparison.

3. Results

3.1. Nutritional parameters

The alcohol treated animals consumed a lower solution volumewhen compared to control, Tween and vitamin E groups. The samebehavior was observed in relation to standard chow diet consump-tion (Table 2).

Animals weight did no differ from each other at the beginningof experiment (p = 0.91), but at the end, the alcohol treated ani-mals’ weight were lower (Table 3, p < 0.05).

3.2. Lipid peroxidation and protein oxidative damage

Lipid peroxidation levels (TBARS) and protein carbonyls areshown in Fig. 1a and b. Rats subjected to alcohol treatment exhibitedlower TBARS levels when compared to control, Tween and vitamin Egroups (Fig. 1a, control = 0.130 ± 0.017, alcohol = 0.084 ± 0.036,Tween = 0.148 ± 0.013, vitamin E = 0.160 ± 0.015, alcohol/vitaminE = 0.108 ± 0.031, p < 0.05). In addition, alcohol intake associatedwith vitamin E co-treatment showed lower TBARS levels when com-pared to vitamin E-treated animals. Protein carbonyls were not sig-nificantly different between groups (p = .554).

3.3. Superoxide dismutase and catalase activities

SOD and catalase activities are shown in Fig. 2a and b, respec-tively. SOD activity was higher in Tween group and lower in alco-hol/vitamin E group when compared with other groups(Tween = 18.49 ± 12.67, vitamin E = 14.90 ± 5.30, alcohol = 10.55 ±4.44, control = 8.74 ± 5.25, alcohol/vitamin E = 6.58 ± 3.33, p <0.05). CAT activity was higher in alcohol/vitamin E group when com-pared to control and vitamin E groups (alcohol/vitaminE = 0.024 ± 0.006, Tween = 0.018 ± 0.006, alcohol = 0.016 ± 0.006,control = 0.014 ± 0.005, vitamin E = 0.011 ± 0.007, p < 0.05). SOD/CAT ratio are shown in Fig. 2c. Alcohol/vitamin E animals presenteda lower SOD/CAT ratio when compared to vitamin E ones (alcohol/vitamin E, mean rank = 6,57; vitamin E, mean rank = 20,67, p < 0.05).

Alcohol 10 6.35 15.15Tween 6 11.56 38.15Vitamin E 6 11.73 34.11Alcohol vitamin

E10 6.48 16.21

Results are expressed as mean.

Table 3Initial and final weight (g) and change in body weight (%) of female Wistar rats in different experimental groups.

Group n Initial weight (g) Final weight (g) Change in body weight (%)

Control 6 230.25 (12.27) NS 232.33 (10.40)a p < 0.05 0.96 (2.42) a p < 0.05Alcohol 10 222.44 (21.57) 196.95 (20.19)b �11.21 B(7.75)b

Tween 6 228.37 (10.95) 229.82 (16.14)a 0.57 (3.19)a

Vitamin E 6 225.65 (9.84) 224.82 (10.89)a �0.37 (1.70)a

Alcohol vitamin E 10 221.27 (19.53) 203.70 (15.00)b �7.48 (9.11)b

Results are expressed as mean ± standard deviation. a,bValues not sharing a common superscript letter differ significantly at p < 0.05 (ANOVA followed by Tukey). NS = notsignificant.

Fig. 1. Oxidative damage parameters in rats tongue mucosa. Values representmeans and SD. Fig. 2. Antioxidant enzymes activities in rats tongue mucosa. Values represent

means and SD.

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4. Discussion

This is the first time that acute alcohol effects upon rats tongueoxidative stress parameters were assessed in literature. It was ob-served a decreased lipid peroxidation level in alcohol-treated ani-mals (Fig. 1a). Animals subjected to alcohol and vitamin Epresent a lipid peroxidation level lower than vitamin E-treated ani-mals (Fig. 1a), which reinforces our hypothesis that alcohol-in-duced lipid peroxidation levels decrease. This result was inagreement with previous studies, where an alcohol antioxidant ef-fect was observed in Sertoli cell cultures and kidney of rat tissuesafter low doses of alcohol administration (Dal-Pizzol et al., 2001;Bertelli et al., 2005, respectively). On the other hand, Comporti etal. (1967), have demonstrated an increase in liver lipid peroxida-tion. These discrepancies in reported data are likely to be linkedto differences in the tissues studied, as well as dose, concentrationand route of alcohol administration (Nordmann et al., 1992). Thedifferences in tissue responses to alcohol are complex and mayvary according to the different tissues involved. The liver and kid-ney are certainly affected by alcohol ingestion earlier than oral mu-cosa, since they are the tissues more involved with alcoholmetabolism and excretion, respectively. The liver is the major siteof alcohol metabolization. Liver alcohol oxidative damage has beenwell demonstrated in the literature (Lieber, 1988; Johnson, 1995).Free radical mechanisms also seem to be implicated in the toxicity

of alcohol to several extrahepatic tissues. Overall, the availabledata concern the gastric mucosa, the central nervous system, theheart, and the testes (Nordmann et al., 1992). In oral mucosa, thereis lower amounts of alcohol degradation, so, at least after short-term alcohol administration, it is reasonable that alcohol effectswas less conspicuous than liver. Therefore, there is a tissue-specificsensitivity; liver, the main site of alcohol metabolism, presentsalcohol damage effects earlier (3 days) (Wang and Cederbaum,2007). In the same way, tissues such as central nervous systemhas been showed to be more sensitive to alcohol damage (Nord-man, 1987; Chu et al., 2007), because alcohol lead to an increasedROS generation and antioxidant capacity decrease (Nordman,1987; Chu et al., 2007). Microsomal Ethanol Oxidizing System(MEOS) is an accessory pathway of alcohol metabolization, involv-ing an enzyme called cytochrome P450 (CYP2E1) which is alcohol-inducible (Lieber, 1997). After chronic ethanol consumption, thereis a 4- to 10-fold induction of CYP2E1, that may lead to ROS gener-ation increase (Lieber, 1997). Antioxidant enzymes activity hasbeen found in oral mucosa and in salivary glands (Yang et al.,2002; Campos et al., 2005), but there are no reports about oxida-tive stress parameters after alcohol consumption in literature.Our hypothesis was that vitamin E could improve antioxidant de-fenses as a non-enzymatic antioxidant (Fig. 3). Vitamin E supple-mentation method was based on Kalender et al. (2004), who

Fig. 3. Theoretical model of vitamin E protective mechanism.

V.C. Carrard et al. / Food and Chemical Toxicology 47 (2009) 1058–1063 1061

demonstrated its protective effects. We intend to use gavage andtwice a week supplementation allow us to maintain vitamin E lev-els, since accumulation can occur on liver.

Rat oral mucosa has a high degree of keratinization that possi-bly provides a relative level of protection when the animals aresubjected to short-term exposure. The keratinized layer of lingualepithelium show extremely flattened and dehydrated cells. It ispossible that high alcohol concentrations improve this protectivelayer, as an effect described as ‘‘fixative” by Squier et al. (1986)and Du et al. (2000) that improve epithelium permeability barrier.Squier et al. (2003) pointed that lipid metabolism alteration due toalcohol is related to permeability barrier impairment (Squier et al.,1986; Du et al., 2000). Those findings may be supported by lipidperoxidation reduction demonstrated in the present study(Fig. 1a). These considerations may be reinforced by lipid granulesaccumulation observed in some studies as a consequence of3 months of alcohol consumption (Mascrès and Joly, 1981; Marti-nez etal., 1998, 2002). Squier et al. (2003) suggested that these per-meability barrier modifications are related to systemic effects ofalcohol on liver metabolism. Since this findings were reported‘‘in vitro” (Squier et al., 1986; Du et al., 2000) and ‘‘in vivo” studies(Howie et al., 2001; Squier et al., 2003), it may be speculated thatalcohol influence lipid metabolism by association with local andsystemic mechanisms.

Previous studies carried out by our research group (Maito et al.,2003; Carrard et al., 2004) have shown that, at least in mice, etha-nol consumption induces increased cell proliferation in the epithe-lium of tongue dorsum. These findings support the hypothesis thatbiochemical alterations could take place in tongue dorsum, evenafter acute consumption. The present study is an early investiga-tion where we intend to evaluate the acute effects of alcohol con-sumption. Further studies focusing on other variables, such as thechronic effects of alcohol and some aspects of the ventral tongueshould be conduced. In a pilot study we observed that even ventraltongue epithelium exhibits keratinization in rats. Taking this con-sideration in mind, it is possible to suggest that the associationsbetween risk of oral cancer and anatomic site observed usingexperimental models based on rodents are probably different fromthe equivalent associations in humans.

Vitamin E animals exhibited higher TBARS levels than control(Fig. 1a). This finding could be attributed to Tween 80, the vehiclefor vitamin E administration.

Higher SOD activity was observed in Tween and in vitamin Egroups when compared to alcohol/vitamin E (Fig. 2a), supportthe pro-oxidative effect attributed to Tween 80, which inducedSOD modulation. In addition, our results suggest that alcohol asso-ciated to vitamin E co-treatment plays a protective role against freeradicals or ROS produced by Tween. The SOD modulation exhibitedby Tween-treated animals was sufficient to scavenge free radicalsor ROS, therefore no alteration was observed in lipid or protein oxi-dation parameters in this animals group (Fig. 1a and b).

Alcohol treatment produced no alterations in CAT activity(Fig. 2b). CAT activity increased in the alcohol/vitamin E group,

when compared to the control and vitamin E animals. This couldbe a controversial finding since alcohol/vitamin E animals showedsimilar SOD activity when compared to control group. However,this finding could be the result of alcohol metabolism, since cata-lase is one of the alternative enzymes for alcohol metabolization.Therefore, this enzyme activity was probably stimulated by alcoholassociated to vitamin E co-supplementation, in order to metabolizethese substances. Since CAT activity increased in this group with-out a proportional increase in SOD activity, deleterious effectsmight be observed after longer treatment periods. This SOD/CATimbalance may reduce the hydrogen peroxide level, which isimportant for cellular signaling in cellular proliferation and differ-entiation. However, in vitamin E group we observed an increasethe SOD/CAT ratio (Fig. 2c). It may be assumed that this alterationrelates to increased hydrogen peroxide levels, resulting in thehigher lipid peroxidation levels observed in these animals (Fig. 1a).

Long-term alcohol intake experiments could change the oxida-tive damage parameters as well as antioxidant enzyme activity,improving the effects of different treatments. The SOD/CAT activityimbalance (Fig. 2c) could have disturbed hydrogen peroxide med-iated cellular signaling in alcohol/vitamin E group and superoxide-mediated signaling in Tween group.

Although some authors consider Tween 80 to be a suitable vehi-cle for the preparation of vitamin E solution (Krishnamurthy andBieri, 1963; Ribeiro et al., 2005; Ohta et al., 2006), the pro-oxidanteffect observed in our results demonstrates that it is inappropriate.In future studies, vitamin E should be dissolved in another vehicle,such as corn oil. Alternatively, a lower concentration of Tweensolution, such as 0.01% could be employed (Campos et al., 2005).In this study, Tween 80 was chosen because it allows for awater-soluble preparation, which was suitable in order to improvegastrointestinal absorption. No studies have reported that Tween80 has noxious effects and many studies have used it as a vehiclefor vitamin E (Losowsky et al., 1972; Sheltaway and Lowosky,1975; Ilavazhagan et al., 2001; Ribeiro et al., 2005). Curiously, wedid observe some noxious effects in Tween groups, which wereunexpected. In order to distinguish between the effects of vitaminE and Tween 80, we included ‘‘Tween 80” and ‘‘vitamin E” groups.Since the aim of the study was not to assess the effects of associa-tion between alcohol and Tween 80, we assumed that to include analcohol/Tween 80 group would be not be contributory.

Unfortunately, since Tween exhibited a pro-oxidant effect, theantioxidant effect of vitamin E observed in other studies (Ilavazh-agan et al., 2001) could not be properly evaluated in this study.Although Kalender et al. (2004) have demonstrated protective ef-fects with the doses employed in the present study (200 mg/kg),it possible that oral tissues require higher doses for vitamin E toconfer protective effects. The method employed for vitamin E sup-plementation was based on the literature. To the best of ourknowledge there are no studies that have defined the optimaldoses to affect oral tissues, so we based our method on a studyin which the protective effects of vitamin E were observed. Somestudies have demonstrated the protective effects of vitamin E usinglower doses (10 mg/kg) given two or three times a week. Evendoses such as 25 IU, administered orally once a week have beenshown to be capable of conferring protective effects (Bascuñan-Castillo et al., 2004). Our intention was to avoid an excessive dos-age, because some studies have showed that higher vitamin Edoses have oxidative effects. We intended to maintain the vitaminE levels at an optimal dosage. If a slightly higher dose was given, acertain quantity of vitamin E could be stored in the liver (Bieri andFarrell, 1976).

In summary, several studies have pointed out that alcohol,when administered both acutely and on a chronic basis, causes oxi-dative stress to most tissues including the liver (Hoek and Pastori-no, 2002; Lieber, 2004), central nervous system (Goodlett and

1062 V.C. Carrard et al. / Food and Chemical Toxicology 47 (2009) 1058–1063

Horn, 2001), skeletal muscle (Mansouri et al., 2001; Adachi et al.,2006) and testes (Grattagliano et al., 1997; Schlorff et al., 1999).

Our alcohol-exposure experimental groups exhibited signals ofmalnutrition (Table 3), observed by impaired weight gain over twoweeks. These could be explained by lower standard diet intake (Ta-ble 2), which is a behavior often related to in animals as well as inhumans that consume alcohol (Seitz and Simanowski, 1988).

Our alcohol administration method somehow reproduced theseaspects of alcoholism. Alcohol treated animals had lower weightgain (Table 3) and present a consequent lower weight at the endof experimental period (Table 3) that may be attributed to lowerchow diet intake. The lower alcohol solution consumption couldbe explained by alcohol unpleasant taste. Diarrhea absence coulddiscard the dehydration possibility.

5. Conclusion

The results of this study suggest that short-term alcohol intakemodify the cellular lipid metabolism. Long period of alcoholadministration should be conducted in order to asses the chroniceffects on rat tongue mucosa. Simultaneous treatment with alcoholand vitamin E induced a higher catalase activity and no oxidativedamage was observed. The protective effect of vitamin E againstROS that could be expected (Fig. 3) was not observed, probably be-cause the vehicle used (Tween 80) exhibited an unknown pro-oxi-dant effects, even though the literature suggested that Tween 80was a good vehicle for vitamin E.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was conducted with the support of a grant receivedfrom CAPES (Brazilian Agency for the Improvement of Higher Edu-cation Personnel) and CNPq (National Council for Scientific andTechnological Development). The authors would like to thank toLaboratório de Histopatologia Prof. Dr. J.J. Barbachan (Patologia Bu-cal), to the Centro de Estudos em Estresse Oxidativo (Laboratório32) at the Departamento de Bioquímica, UFRGS, as well as CarenBavaresco, Patrícia Sesterheim, Luiza Braga, Isabel da Silva Lauxen,Cristiano Susin Luciana Adolfo, Leandro Nunes, Adriana Aguiar andChristiane Gerhard for technical support and Marcos Schwengberfor assistance with surgical procedures.

References

Adachi, J., Kudo, R., Asano, M., Ueno, Y., Hunter, R., Rajendram, R., et al., 2006.Skeletal muscle and liver oxysterols during fasting and alcohol exposure.Metabolism 55 (1), 119–127.

Aebi, H., 1984. Catalase in vitro. Meth. Enzymol. 105, 121–126.Bieri, J.G., Farrell, P.M., 1976. Vitamin E. Vitam. Horm. 34, 31–75.Bascuñan-Castillo, E.C., Erickson, R.P., Howison, C.M., Hunter, R.J., Heidenreich, R.H.,

Hicks, C., et al., 2004. Tamoxifen and vitamin E treatments delay symptoms inthe mouse model of Niemann–Pick C. J. Appl. Genet. 45 (4), 461–467.

Bertelli, A.A., Migliori, M., Filippi, C., Gagliano, N., Donetti, E., Panichi, V., et al., 2005.Effect of ethanol and red wine on ochratoxin A-induced experimental acutenephrotoxicity. J. Agric. Food Chem. 53 (17), 6924–6929.

Campos, S.C., Moreira, D.A., Nunes, T.D., Colepicolo, P., Brigagão, M.R., 2005.Oxidative stress in alcohol-induced rat parotid sialadenosis. Arch. Oral Biol. 50(7), 661–668.

Carrard, V.C., Filho, M.S., Rados, P.V., Chaves, A.C., Lauxen Ida, S., 2004.Quantification of silver-staining nucleolar organizer region in epithelial cellsof tongue of mice after exposure to, or intake of, alcohol. Alcohol 34 (2–3), 233–238.

Chu, J., Tong, M., de la Monte, S.M., 2007. Chronic ethanol exposure causesmitochondrial dysfunction and oxidative stress in immature central nervoussystem neurons. Acta Neuropathol. 113, 659–673.

Comporti, M., Hartman, A., Di Luzio, N.R., 1967. Effect on in vivo and in vitro ethanoladministration on liver lipid peroxidation. Lab. Invest. 16 (4), 616–624.

Dal-Pizzol, F., Klamt, F., Dalmolin, R.J., Bernard, E.A., Moreira, J.C., 2001. Mitogenicsignaling mediated by oxidants in retinol treated Sertoli cells. Free Radical Res.35 (6), 749–755.

Das, S.K., Vasudevan, D.M., 2007. Alcohol-induced oxidative stress. Life Sci. 81, 177–187.

Dey, A., Cederbaum, A.I., 2006. Alcohol and oxidative liver injury. Hepatology 43 (2Suppl. 1), S63–S74.

Du, X., Squier, C.A., Kremer, M.J., Wertz, P.W., 2000. Penetration of N-nitrosonornicotine (NNN) across oral mucosa in the presence of ethanol andnicotine. J. Oral Pathol. Med. 29 (2), 80–85.

Goodlett, C.R., Horn, K.H., 2001. Mechanism of alcohol-induced damage to thedeveloping nervous system. Alcohol Res. Health 25 (3), 175–182.

Grattagliano, I., Vendemiale, G., Errico, F., Bolognino, A.E., Lillo, F., Salerno, M.T.,Altomare, E., 1997. Chronic ethanol intake induces oxidative alterations in rattestis. J. Appl. Toxicol. 17 (5), 307–311.

Halliwell, B., 1991. Drug antioxidant effects: a basis for drug selection? Drugs 42 (4),569–605.

Hoek, J.B., Pastorino, J.G., 2002. Ethanol, oxidative stress, and cytokine-induced livercell injury. Alcohol 27 (1), 63–68.

Howie, N.M., Trigkas, T.K., Cruchley, A.T., Wertz, P.W., Squier, C.A., Williams, D.M.,2001. Short-term exposure to alcohol increases the permeability of human oralmucosa. Oral Dis. 7 (6), 349–354.

Ilavazhagan, G., Bansal, A., Prasad, D., Thomas, P., Sharma, S.K., Kain, A.K., Kumar, D.,Selvamurthy, W., 2001. Effect of vitamin E supplementation on hypoxia-induced oxidative damage in male albino rats. Aviat. Space Environ. Med. 72(10), 899–903.

Johnson, P.J., 1995. Acute and chronic liver disease. In: Marshall, W.J., Bangert, S.K.(Eds.), Clinical Biochemistry – Metabolic and Clinical Aspects. ChurchillLivingstone, New York, pp. 243–245.

Kalender, S., Kalender, Y., Ogutcu, A., Uzunhisarcikli, M., Durak, D., Acikgoz, F., 2004.Endodulfan-induced cardiotoxicity and free radical metabilism in rats: theprotective effect of vitamin E. Toxicology 202 (3), 227–235.

Krishnamurthy, S., Bieri, J.G., 1963. The absorption, storage, and metabolism of a-tocopherol-C14 in the rat and chicken. J. Lipid Res. 4, 330–336.

Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W.,Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content inoxidatively modified proteins. Meth. Enzymol. 186, 464–478.

Lieber, C.S., 1988. Biochemical and molecular basis of alcohol-induced injury to liverand other tissues. New Engl. J. Med. 319 (25), 1639–1650.

Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clin. Chim. Acta257 (1), 59–84.

Lieber, C.S., 2004. Alcoholic fatty liver: its pathogenesis and mechanism ofprogression to inflammation and fibrosis. Alcohol 34 (1), 9–19.

Losowsky, M.S., Kelleher, J., Walker, B.E., Davies, T., Smith, C.L., 1972. Intake andabsorption of tocopherol. Ann. NY Acad. Sci. 203, 212–222.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275.

Maito, F.L., Rados, P.V., Filho, M.S., Barbachan, J.J., Quadros, O., 2003. Proliferatingcell nuclear antigen expression on tongue of mice after intake of, or topicalexposure to, alcohol. Alcohol 31 (1–2), 1–6.

Mansouri, A., Demeilliers, C., Amsellem, S., Pessavre, D., Fromenty, B., 2001. Acuteethanol administration oxidatively damages and depletes mitochondrial DNA inmouse liver, brain, heart, and skeletal muscles: protective effects ofantioxidants. J. Pharmacol. Exp. Ther. 298 (2), 737–743.

Martinez, M., Martinez, F.E., da Cunha, M.R., Segatelli, T.M., Pinheiro, P.F., Almeida,C.C., 2002. Morphological effects on the hard palatine mucosa of Calomyscallosus submitted to experimental chronic alcoholism. J. Submicrosc. Cytol.Pathol. 34 (1), 77–83.

Martinez, M., Martinez, F.E., Watanabe, I., 1998. Morphological changes on the hardpalatine mucosa of rats (Rattus norvegicus albinus) after chronic alcoholconsumption. J. Submicrosc. Cytol. Pathol. 30 (3), 379–384.

Mascrès, C., Joly, J.G., 1981. Histochemical and ultrastructural study of the rat oralmucosa, after chronic administration of alcohol. J. Biol. Buccale 9, 279–295.

McDonough, K.H., 2003. Antioxidant nutrients and alcohol. Toxicology 189 (1–2),89–97.

McMillen, B.A., Crawford, M.S., Kulers, C.M., Williams, H.L., 2005. Effects of ametabotropic, MGLU5, Glutamate receptor antagonist on ethanol consumptionby genetic drinking rats. Alcohol Alcohol. 40 (6), 494–497.

Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autoxidation ofepinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247(10), 3170–3175.

Neves, M.M., Borges, D.R., Vilela, M.P., 1989. Concentração de etanol nas bebidasmais consumidas no Brasil. Gastroenterol. Endosc. Dig. 8, 17–20.

Nordman, R., 1987. Oxidative stress from alcohol in the brain. Alcohol Alcohol. 1(Suppl. 1), 75–82.

Nordmann, R., Ribière, C., Rouach, H., 1990. Ethanol-induced lipid peroxidation andoxidative stress in extrahepatic tissues. Alcohol Alcohol. 25, 231–237.

Nordmann, R., Ribière, C., Rouach, H., 1992. Implication of free radical mechanismsin ethanol-induced cellular injury. Free Radical Biol. Med. 12 (3), 219–240.

Ohta, A., Kobayashi, T., Imai, Y., Inui, K., Yoshino, J., Nakazawa, S., 2006. Effect of oralvitamin E administration on acute gastric mucosal lesion progression in ratstreated with compound 48/80, a mast cell degranulator. Biol. Pharm. 29 (4),675–683.

V.C. Carrard et al. / Food and Chemical Toxicology 47 (2009) 1058–1063 1063

Ribeiro, M.C.P., de Ávila, D.S., Schneider, C.Y.M., Hermes, F.S., Furian, A.F., Oliveira,M.S., et al., 2005. a-Tocopherol protects against pentylenetetrazol- andmethylmalonate-induced convulsions. Epilepsy Res. 66 (1–3), 185–194.

Schlorff, E.C., Husain, K., Somani, S.M., 1999. Dose and time dependent effects ofethanol on antioxidant system in rat testes. Alcohol 18 (2–3), 203–214.

Seitz, H.K., Simanowski, U.A., 1988. Alcohol and carcinogenesis. Annu. Rev. Nutr. 8,99–119.

Sheltaway, M.J., Lowosky, M.S., 1975. Effect of non-ionic detergents on theabsorption of the fat and alpha-tocopherol in the rat. Nutr. Metab. 18 (5–6),267–271.

Squier, C.A., Cox, P., Hall, B.K., 1986. Enhanced penetration of nitrosonornicotineacross oral mucosa in the presence of ethanol. J. Oral Pathol. Med. 15 (5), 276–279.

Squier, C.A., Kremer, M.J., Wertz, P.W., 2003. Effect of ethanol on lipid metabolismand epithelial permeability barrier of skin and oral mucosa in the rat. J. OralPathol. Med. 32, 595–599.

Valenzuela, A., 1991. The biological significance of malondialdehyde determinationin the assessment of tissue oxidative stress. Life Sci. 48 (4), 301–309.

Wang, X., Cederbaum, A., 2007. Acute ethanol pretreatment increases FAS-mediatedliver injury in mice. Role of oxidative stress and CYP2E1-dependent and -independent pathways. Free Radical Biol. Med. 42 (7), 971–984.

Wu, D., Cederbaum, A.I., 2003. Alcohol, oxidative stress, and free radical damage.Alcohol Res. Health 27 (4), 277–284.

Yang, J., Lamm, E.W., Hammad, H.M., Oberley, T.D., Oberley, L.W., 2002. Antioxidantenzyme levels in oral squamous cell carcinoma and normal human oralepithelium. J. Oral Pathol. Med. 31 (2), 71–77.