Abstract
Context: Alcohol consumption has been related to a cell proliferation increase in oral epithelium but its mechanism remains unclear.
Objective: The aim of this study was to investigate whether oxidative stress parameters are implicated in the induction of cell proliferation in rat tongue epithelium after different times of chronic alcohol consumption.
Materials and methods: Cell proliferation was assessed in tongue epithelium using AgNOR (argyrophilic proteins related to active nucleolar organizer regions) quantification. Oxidative stress parameters [lipid peroxidation, protein carbonyls, superoxide dismutase activity and catalase (CAT) activity and immunocontent] and Nrf2 immunocontent were quantified in tongue homogenates.
Results and discussion: Mean AgNOR numbers (mAgNOR) per nucleus was 2.22 ± 0.30 in ventral tongue epithelium after 120 days of alcohol consumption (vs. 1.87 ± 0.18 for control animals and 1.91 ± 0.23 for animals treated with alcohol for 60 days) indicating cell proliferation increase (p < 0.05, ANOVA followed by Tukey post hoc). Interestingly, 60 days of alcohol consumption induced changes in oxidative stress parameters, but no alteration in cell proliferation. Vitamin E co-treatment was conduced in order to evaluate its possible protective effects. The 120 day Tween + vitamin E + alcohol treatment induced an increase in mAgNORs when compared to the Tween + vitamin E treated group (respectively 2.10 ± 0.30 vs. 1.77 ± 0.11, p < 0.05, ANOVA followed by Tukey post hoc), showing that vitamin E co-treatment had no protective effects. In addition, an inverse association was observed between CAT activity and AgNORs quantity (R = −0.32; p < 0.05, Person’s correlation) as well as the possible involvement of Nrf2 in alcohol-related damage.
Conclusions: Our findings suggest that the increase in cell proliferation associated with alcohol-related damage has no direct relation with an imbalance in oxidative parameters. In contrast, our results indicate that hydrogen peroxide may be implicated in cellular signaling during proliferation in the oral mucosa.
Introduction
Alcohol acts as an independent risk factor for oral cancer (CitationFioretti et al., 1999; CitationMoreno-López et al., 2000; CitationBagnardi et al., 2001; CitationGoldstein et al., 2010). Some studies demonstrated that there is a relation with dose and/or duration of alcohol intake (CitationFioretti et al., 1999; CitationBagnardi et al., 2001; CitationGoldstein et al., 2010), but its mechanism of action remains unclear (CitationWight & Ogden, 1998; CitationCarrard et al., 2004; CitationGoldstein et al., 2010). Increase in epithelial proliferation (CitationValentine et al., 1985; CitationMaier et al., 1994; CitationMaito et al., 2003; CitationCarrard et al., 2004) and higher permeability to tobacco carcinogens (CitationSquier et al., 1986; CitationDu et al., 2000) have been demonstrated in oral mucosa after alcohol exposure. These findings support the hypothesis that alcohol induces alterations in oral mucosa, which could be related to oral cancer, since increase in cell proliferation is one of the early stages in carcinogenesis.
Oxidative stress is the imbalance between the production of reactive oxygen substances (ROS) and the capacity of antioxidants to respond (CitationWu & Cederbaum, 2003). In these situations, ROS can react with and damage complex cellular molecules such as lipids, proteins, and DNA (CitationWu et al., 2006). This phenomenon could be one of the mechanisms associated with the alcohol-mediated increase in cell proliferation rates (CitationValentine et al., 1985; CitationMaito et al., 2003; CitationCarrard et al., 2004) as well as the risk of oral cancer attributed to alcohol (CitationGuyton & Kensler, 1993; CitationKlaunig & Kamendulis, 2004). A previous study (CitationCarrard et al., 2009) suggested that even a short term exposure to alcohol induced biochemical disturbances in oral mucosa of rats. In addition, Nrf2, a redox-sensitive transcription factor involved in antioxidant defenses which is usually associated to the response to xenobiotics (CitationMotohashi & Yamamoto, 2004), could be involved in the pathogenesis of alcohol-induced cell and tissue damage. CitationVincon et al. (2003) demonstrated that vitamin E supplementation attenuates the increased epithelial proliferation in rat colon mucosa due to alcohol intake. This could be associated with antioxidant properties of vitamin E. However, the mechanisms relating chronic alcohol consumption and cell proliferation increase in oral epithelial cells remains unknown. Since ROS may act on cellular proliferation signaling (CitationKamata et al., 1999; CitationGenestra, 2007) oxidative stress parameters could be related to cell proliferation rate in oral epithelium.
AgNORs (argyrophilic proteins related to active nucleolar organizer regions) quantification is used as a proliferation marker that provides information about the velocity of cell proliferation (cell proliferation rate) during the cell cycle (CitationDerenzini et al., 2000; CitationSirri et al., 2000). AgNORs are related to proteins involved in rRNA (ribosomal ribonucleic acid) synthesis, which can be visualized as black dots in the nucleus using a silver staining technique (CitationPloton et al., 1986). This histochemical technique is different from immunohistochemistry, which indicates only if cells are undergoing division (cell growth fraction) (CitationDerenzini et al., 2000; CitationSirri et al., 2000).
The aim of the present study was to evaluate whether alterations in oxidative stress parameters and Nrf2 immunocontent could be involved in the mechanisms of epithelial cell proliferation in rat tongue after different times of chronic alcohol consumption.
Materials and methods
Animals and treatment
A total of 48 female Wistar rats, three months of age and weighting 190–260 g, were housed in a temperature-controlled room (24°C ± 1°C) and 12:12 h reverse light/dark cycles. This study was approved by the Ethics Committee in Animals Research of the Universidade Federal do Rio Grande do Sul.
Animals were assigned to one of the following six groups using a weighted stratified randomization method:
C = control (6 animals)
ALC 60 = alcohol/60 days (10 animals)
ALC 120 = alcohol/120 days (10 animals)
T = Tween (6 animals)
T+VE = Tween + vitamin E (6 animals)
T+VE+ALC 120 = Tween + vitamin E + alcohol/120 days E (10 animals)
Animals in the C, T and T + VE groups were fed with a standard laboratory chow diet for rodents (Nuvilab/CR1, Nuvital Nutrientes LTDA, Colombo, Paraná, Brazil) and tap water ad libitum. The other experimental groups (ALC 60, ALC 120 and T+VE+ALC 120) received the same standard diet, but water was replaced with 40% (v/v) ethyl alcohol throughout the experiment. The 40% alcohol concentration was chosen because it is the same concentration found in “cachaça,” a kind beverage most frequently consumed by the Brazilian population (CitationNeves et al., 1989). During the first week, alcohol concentration was increased gradually from 5 to 40% (adapted from CitationMcMillen et al., 2005). After 60 days, 40% ethyl alcohol was replaced with water in ALC 60, gradually decreasing the alcohol concentration from 40 to 0% in one week.
Vitamin E (α-tocopherol dissolved in 5% Tween 80 solution) was administered orally by gavage [200 mg/kg, twice a week (CitationKalender et al., 2004)] to rats in the T+VE+ALC 120 and T+VE groups. C, ALC 60 and ALC 120 animals received saline by gavage. Animals in the T received 5% Tween 80 solution (CitationKrishnamurthy & Bieri, 1963) at the same dose as animals in the T+VE and T+VE+ALC 120 groups to evaluate the effect of vitamin E vehicle.
After 120 days, animals were killed by asphyxiation in a CO2 chamber, and the tongue was surgically removed.
Cell proliferation measurement
One half of the tongue of all specimens was fixed in 10% neutral buffered formalin for 24 h and transversally embedded in paraffin so that all the epithelial layers and the connective tissue could be visualized. Two 4-µm sections were cut from each specimen: one was hematoxylin and eosin-stained, and the other was silver-stained for visualization of AgNORs according to the method described by CitationPloton et al. (1986).
Images of microscopic fields of all slides were captured with a Sony Digital Video Camera (DSC W50, Sony Co., Tokyo, Japan) connected to a binocular microscope Zeiss-Standard 20 (Carl Zeiss Inc., Thornwood, NY, USA) at 400 × magnification, 3 Mpx resolution and standardized optical zoom.
Dorsal and ventral tongue epithelia of each slide were analysed and images were recorded for 30 consecutive microscopic fields or for all the microscopic fields when there were fewer than 30 (CitationCarrard et al., 2004).
AgNOR dots per nucleus were counted using the manual count tool. On each slide 200 cells were assessed – 100 basal layer cells and 100 suprabasal layer cells from each anatomic site. Microscopic fields with superimposed cells and areas of artifact were excluded. Dots that could not be distinguished from each other were counted as a single dot, following the standardized counting method described by CitationCrocker et al. (1989). In the granular cell layer, lysosomal acid phosphatase activity was indicated by silver granules (CitationMascrès & Joly, 1981); the areas with this type of staining were not included in this study (CitationCarrard et al., 2004). AgNORs count was recorded as mean AgNOR number per nucleus (mAgNOR) and percentage of cells with more than 1 AgNOR per nucleus (pAgNOR > 1).
Measurement procedures were adjusted before evaluation, and the same procedures were repeated one week later (paired Student t-test, p = 0.86). A blind examiner performed the analysis of the slides.
Evaluation of oxidative stress parameters
A third of the other half of the tongue was homogenized in 1000 µL of ice-cold phosphate buffered saline (PBS, pH 7.4), sonicated in (4 cycles of 10 s) ice bath and stored at −80°C for further analyses.
Lipid peroxidation
The formation of TBARS (thiobarbituric acid reactive substances) during an acid-heating reaction (CitationValenzuela, 1991) was used as an index of lipid peroxidation. Briefly, 300 µL of samples were mixed with 600 µL of 15% trichloroacetic acid and 500 µL of 0.67% thiobarbituric acid, and heated in a boiling water bath for 20 min. TBARS were determined by the absorbance at 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. Results were expressed as malondialdehyde equivalents per milligram of protein.
Quantification of protein carbonyls
The oxidative damage to protein was quantified by the determination of carbonyl groups according to their reaction with dinitrophenylhydrazine (DNPH) using a slightly modified version of the method described by CitationLevine et al. (1990). Proteins were precipitated by the addition of 20% trichloroacetic acid and reacted with DNPH. After that, 8 M urea was added, and carbonyl contents were quantified by determining the absorbance at 370 nm using a molar absorption coefficient of 22 000 M−1
Catalase and superoxide dismutase activities
To determine catalase (CAT) activity, tissue portions were sonicated in 50 mM phosphate buffer, and the resulting suspension was centrifuged at 6200g for 10 min. The supernatant was used for the enzyme assay. CAT activity was quantified by the rate of decrease in hydrogen peroxide (10 mM) absorbance at 240 nm (CitationAebi, 1984). Superoxide dismutase (SOD) activity was assayed by quantifying the inhibition of adrenaline self-oxidation absorbance at 480 nm (CitationMisra & Fridovich, 1972).
Protein quantification
All the results were normalized to protein content (CitationLowry et al., 1951).
Immunoblot (CAT and Nrf2)
Tongue homogenates were lysed in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 1% [w/v] SDS, 10% [v/v] glycerol) and an equal amount of total protein (60 mg/well) was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Protein loading and electroblotting efficiency were evaluated using Ponceau S staining, and the membrane was blocked in Tween-Tris buffered saline (TTBS: 100 mM Tris-HCl, pH 7.5, containing 0.9% NaCl and 0.1% Tween-20) with 5% albumin and incubated overnight with the primary antibody to be tested. The membrane was washed, and the immunoreactivity was detected by enhanced chemiluminescence. Densitometric analysis of the films was performed with the IMAGE J® software (available on http://rsb.info.nih.gov/ij/index.html); the spots corresponding to CAT and NrF2 (nuclear factor erythroid-2 related factor 2: redox-sensitive transcription factor) were normalized using β-actin as an internal control.
Correlation between cell proliferation rate and oxidative parameters
The correlation between cell proliferation rate and oxidative parameters was assessed because ROS may be involved in cellular signaling for cell proliferation. Cell proliferation parameters were mAgNOR and pAgNOR > 1 (CitationXie et al., 1997).
Statistical analysis
Analysis of variance (ANOVA) was used to compare means between groups when data followed a normal distribution. Tukey’s method was used for multiple comparisons (post hoc test). When data did not follow a normal distribution Kruskal–Wallis followed by Dunn’s post hoc test was used. The results were considered statistically significant if the p value was <0.05. The data are shown in graphs as mean values, standard deviations and mean rank. Pearson’s correlation coefficient was used to test the correlation between oxidative stress parameters and cell proliferation.
Results
Animal body weight at the end of experiment and weight gain percentage used to control the effect of diet, were not statistically different between experimental groups ().
Table 1. Animals body weight (g) and weight gain (%) at the end of experiment.
Cell proliferation rate ( and )
mAgNOR in suprabasal layer cells of ventral tongue epithelium increased in ALC 120 animals when compared with C and ALC 60 animals, and in the T + VE + ALC 120 animals when compared with animals in the T + VE group ().
Figure 1. (A) Cell proliferation rate in suprabasal layer cells of ventral tongue mucosa, *p < 0.05 when compared to C and ALC 60 and *p < 0.05 when compared to T + VE, ANOVA followed by Tukey post hoc; (B) Photomicrograph of ventral tongue epithelium of rat (Tween group) stained to assess the rate of cell proliferation. k – keratin layer; e – epithelial tissue; c – fibrous connective tissue; basal layer cells (arrows) and suprabasal layer cells (asterisk). AgNOR technique, original magnification, ×400.
mAgNOR was lower in the suprabasal layer cells of ventral tongue epithelium ( and ) in the ALC 60 when compared to ALC 120 group (C = 1.87 ± 0.18, ALC60 = 1.91 ± 0.23 and ALC120 = 2.22 ± 0.30). Since pAgNOR showed similar results, it was not shown.
Animals in the ALC 60, ALC 120 and T + VE + ALC 120 groups showed no statistically significant differences in mAgNOR in basal () and in suprabasal layer cells () of dorsal tongue epithelium, or in basal layer cells of ventral tongue epithelium ().
Table 2. mAgNOR values in basal layer cells of rats dorsal tongue epithelium.
Table 3. mAgNOR values in suprabasal layer cells of rats dorsal tongue epithelium.
Table 4. mAgNOR values in basal layer cells of rats ventral tongue epithelium.
Oxidative parameters ()
Lipid peroxidation ()
No differences were found when comparing animals in ALC 120 or T + VE + ALC 120 groups. In the ALC 60 group, TBARS levels decreased (p < 0.05, ANOVA) when compared to the C and with the T + VE + ALC 120 groups.
Figure 2. Lipid peroxidation (A) and carbonyl groups (B) in tongue tissues. The values are means ± SD (*p < 0.05 when compared to C and T + Ve + ALC 120, ANOVA followed by Tukey post hoc).
Figure 3. Antioxidant enzyme activity in tongue tissue of rats in the ALC 60, ALC 120 and T + VE + ALC 120 groups. (A) Superoxide dismutase activity – SOD; (B) Catalase activity – CAT; (C) SOD/CAT ratio. The values are means ± SD for CAT and mean rank for SOD and SOD/CAT ratio (*p < 0.05 when compared to other groups, ANOVA followed by Tukey post hoc; **p < 0.05 when compared to C and T + VE + ALC 120; ***p < 0.05 when compared to ALC 120 group, Kruskal–Wallis followed by Dunn’s test).
Figure 4. Representative immunoblots with respective densito-metric analyses showing CAT (A) and Nrf2 (B) immunocontent in tongue tissues. Data normalized to β-actin content are expressed as means ± S.E. for three individual experiments. n = 3 (*p < 0.05 when compared to others, ANOVA followed by Tukey post hoc).
Protein carbonyls ()
No effects on protein carbonyls were observed in the ALC 60, ALC 120 and T + VE + ALC 120 groups (p = 0.10, ANOVA).
Superoxide dismutase activity ()
ALC 60 animals showed a decrease in the activity of SOD when compared to other groups (p < 0.05, ANOVA).
Catalase activity and immunocontent ( and )
The results showed an increase in CAT activity in ALC 60 group (p < 0.05, ANOVA) and it was also observed that ALC 120 presented similar levels when compared to control and this attenuation was more remarkable with Vitamin E co-treatment (). The treatments with Tween and/or alcohol were noxious to CAT immunocontent ().
SOD/CAT ratio ()
The ALC 60 group showed a lower SOD/CAT ratio (p < 0.05, ANOVA) when compared to the ALC 120 group values ().
Nrf2 immunocontent ()
After the observation of the decrease in CAT immunocontent in the ALC 60 and ALC 120 groups, we decided to evaluate Nrf2, a CAT transcription factor that is overexpressed in response to noxious agents. ALC 60 and ALC 120 groups decreased Nrf2 immunocontent (). Vitamin E co-treatment attenuated alcohol-related Nrf2 decrease. The vitamin E group also showed a reduction in Nrf2, but treatment with Tween 80 alone attenuated this effect.
Correlation between cell proliferation rate and oxidative parameters ()
To verify which oxidative parameters were the source of changes in cell proliferation rate due to alcohol consumption, the Pearson correlation test was used, and results showed that CAT activity was inversely correlated with cell proliferation rate according to the analysis of pAgNOR (R = −0.32; p < 0.05, ANOVA). Lipid peroxidation levels (TBARS, p = 0.58, ANOVA), carbonyl groups (p = 0.68, ANOVA) and SOD activity (p = 0.34, ANOVA) did not show any correlation with cell proliferation rates. Analysis of mAgNOR showed no association with any oxidative parameters (data not shown).
Figure 5. Correlation between cell proliferation rate (pAgNOR) and oxidative stress parameters in suprabasal layer cells in dorsal tongue epithelium of rats (Pearson’s correlation coefficient, p < 0.05).
Discussion
Evaluation of cell proliferation rate
Several studies support evidence that cell proliferation increases in oral epithelium after chronic alcohol intake (CitationValentine et al., 1985; CitationMaier et al., 1994; CitationMaito et al., 2003; CitationCarrard et al., 2004). However, the exact mechanism implicated in such an increase is yet to be investigated (CitationWight & Ogden, 1998; CitationCarrard et al., 2004; CitationGoldstein et al., 2010). To our knowledge, this is the first study assessing the mechanisms of alcohol-related increase in cell proliferation in oral epithelium.
This study focused on the tongue because it is one of the anatomic sites with the highest risk for the development of oral cancer (CitationBoffetta et al., 1992; CitationTurati et al., 2010).
Dorsal tongue epithelium
The analysis of cell proliferation rates revealed no differences between groups in basal and suprabasal layer cells of dorsal tongue epithelium. This finding differs from those reported in previous studies, which found higher proliferative activity in the suprabasal layer cells of dorsal tongue epithelium (CitationValentine et al., 1985; CitationMaier et al., 1994; CitationMaito et al., 2003; CitationCarrard et al., 2004). This difference could be explained by the models used (rats instead of mice), since different animal models appear to have differential sensitivity to alcohol (CitationTabakoff & Hoffman, 2000). Another possible reason is that the present study conducted a shorter intake period, comparing to studies that showed changes in dorsal tongue epithelium (CitationMaito et al., 2003; CitationCarrard et al., 2004).
Ventral tongue mucosa
Basal layer cells of epithelium did not show changes in cell proliferation rates after treatment with ALC 60, ALC 120, or T + VE + ALC 120. However, the analysis of suprabasal layer cells revealed an increase in the number of AgNORs per nucleus that indicated an increase in cell proliferation rate after ALC 120 treatment. This finding corroborates the results of studies that found an increased cell proliferation in the tongue epithelium of mice after longer intake periods (CitationMaito et al., 2003; CitationCarrard et al., 2004). CitationValentine et al. (1985) found an increase in the thickness of the proliferative compartment of the tongue epithelium in humans that usually consume alcohol.
Based on these findings we can suggest that ventral tongue mucosa is more susceptible to alcohol damage. CitationLederman (1964) mentioned that alcohol had a stronger effect on structures belonging to the “food channel” and “reservoir” systems. A lower grade of keratinization and a longer contact with the ingested alcohol may also contribute to this effect.
CitationVincon et al. (2003) reversed the increase in alcohol-related proliferation in the colon mucosa of rats by means of vitamin E supplementation. In our study, the protective effects of vitamin E were found only in terms of attenuation of alcohol-related decrease in Nrf2 immunocontent. Maybe the action of vitamin E varies according to the anatomic site, or the dose used in the present study was not enough to achieve optimal vitamin E levels in tongue tissues. Vitamin E modulates the rate of cell proliferation in several different ways according to the cell type. The molecular basis of sensitivity to vitamin E remains unclear and therefore a different signaling pathway may be used for proliferation in different cell types, or maybe vitamin E transport and metabolism vary according to the cell type (CitationAzzi et al., 1993). Finally, binding proteins (CitationNalecz et al., 1992) associated with vitamin E inhibition could be present in some cells but not in others. CitationChatelain et al. (1993) reported that vitamin E may inhibit protein kinase C, an enzyme that plays a role in proliferation signaling pathways, but their study was performed in smooth muscle cells and not in oral epithelium.
Our evaluation of cell proliferation rate revealed a remarkable finding in relation to the duration of alcohol intake. This finding is in accordance with some studies (CitationFioretti et al., 1999; CitationTurati et al., 2010), but this association is controversial (CitationGoldstein et al., 2010). Additionally, taking the oxidative parameter analyses into account, it can be suggested that the damage in oral mucosa is cumulative and progressive. Our findings may only support the hypothesis that alcohol acts as a promoter, and not as an initiator, of oral cancer (CitationMufti, 1998). Nevertheless, the increase in cell proliferation may be the first step in oral carcinogenesis, since fast turnover tissues are more susceptible to DNA duplication errors (CitationMelnick et al., 1993; CitationTomatis, 1993). Mechanisms responsible for the increase in cell proliferation rates may involve cell proliferation signaling pathways, an adaptive response to external stimulation, or both (CitationPreston-Martin et al., 1993). This mitogenic effect of alcohol may be only an adaptative response to external injuries which induce an increase of desquamation or a signal of initial stages of carcinogenesis, probably related to acetaldehyde, a toxic metabolite produced by alcohol metabolism.
Oxidative parameters
Lipid peroxidation and oxidative protein damage
Animals in the ALC 120 and in the T + VE + ALC 120 groups did not show any changes in TBARS levels. In the ALC 60 group, TBARS levels decreased (), probably due to reduced ROS levels, which could be a consequence of the increase in CAT activity (). These changes in oxidative parameters may be potentially harmful because basal levels of ROS are required for proper cell signaling (CitationKamata & Hirata, 1999; CitationGenestra, 2007). A previous study (CitationCarrard et al., 2009) demonstrated that a short-term exposure to alcohol (14 days) is capable of reducing the levels of TBARS levels in oral mucosa tissues (CitationCarrard et al., 2009). In contrast, tissues from liver (CitationEke et al., 1996), testes (CitationNordmann et al., 1990), kidney (CitationPari & Suresh, 2008) and central nervous system (CitationEmre et al., 2007) have been reported to have an increase in lipid peroxidation levels after the treatment with alcohol. These controversial data could be attributed to differences in the tissue metabolism and sensitivity to alcohol.
Superoxide dismutase activity
In the same way, the effects of alcohol on SOD activity vary according to the tissue studied, as well as to dose, concentration and method of alcohol administration (CitationSchlorff et al., 1999; CitationJurczuk et al., 2004). ALC 60 treatment was associated with lower SOD activity, which indicates a noxious effect probably related to ROS generation.
CAT activity, CAT and Nrf2 immunocontent
Treatment with alcohol decreased Nrf2 immunocontent and led to a decrease in CAT immunocontent. However, at 60 days of alcohol treatment, CAT activity increased, suggesting that the mechanism involved is probably Nrf2-independent. It is well known that CAT activity is not necessarily associated with CAT immunocontent. CAT activity may be modulated by a different mechanism, such as substrate concentration or allosteric activators, and not only by enzyme concentration (CitationSmith et al., 2004). Some studies showed that microsomal oxidizing-ethanol system (MEOS), which is an accessory and adaptative route of alcohol metabolism, generates hydrogen peroxide (CitationMello et al., 2008). The increase of hydrogen peroxide levels probably is one of the pathways involved in the increase in CAT activity found in the ALC 60. Another possibility is that alcohol itself induces an increase in CAT activity, since CAT is an alternative pathway of alcohol metabolization (CitationRiveros-Rosas et al., 1997).
To the best of our knowledge, the Nrf2 involvement as a protective pathway has not been demonstrated elsewhere in oral tissues. Nrf2 may regulate the expression of antioxidant enzymes (CitationEggler et al., 2005; CitationCederbaum, 2009), cell proliferation (CitationHomma et al., 2009) and epithelium keratinization (CitationMotohashi & Yamamoto, 2004). Our data support that alcohol treatment for 60 days induced an increase in CAT activity and the treatment for 120 days increased epithelial proliferation by pathways which are not related to Nrf2 regulation. Clearly, alcohol consumption results in lower Nrf2 immunocontent, which is potentially noxious to cell function. Considering these findings, decrease in epithelial keratinization and/or differentiation observed by CitationValentine et al. (1985) due to alcohol consumption could probably be explained by Nrf2 decrease.
Additionally, the lack of protective properties of Nrf2 induced by chronic alcohol consumption may improve the sensitivity to carcinogens and DNA damage (CitationMotohashi & Yamamoto, 2004). It is likely that Nrf2 may be one of the mechanisms by which a synergistic effect between alcohol and smoke occurs (CitationRamos-Gomez et al., 2001; CitationIizuka et al., 2005). Another important finding of the present study was attenuation of the decrease in Nrf2 immunocontent observed in the T + VE + ALC 120 group, which is in accordance with other studies reporting an induction in Nrf2 after supplementation of other chemopreventive compounds (CitationNa & Surh, 2008).
In summary, the protective mechanisms initially led to CAT induction and protective properties as a consequence. Longer periods of alcohol consumption triggered signaling cascades increasing cell proliferation. However, these mechanisms are complex and only partially described, requiring further studies in order to allow a greater understanding.
SOD/CAT ratio
It was observed that the ALC 60 group showed a decrease in SOD/CAT ratio imbalance and in Nrf2 immunocontent which may explain the reduction on TBARS levels. It is important to emphasize that basal levels of ROS are necessary to promote the turnover of membrane lipids as a physiological process (CitationDröge, 2002).
Correlation of cell proliferation and oxidative parameters
The evaluation of the suprabasal cell layer of the ventral tongue epithelium revealed an important correlation between cell proliferation rate and CAT activity. Our findings indicate that the signaling for cell proliferation may occur due to hydrogen peroxide stimulation in this tissue, corroborating findings from other studies (CitationKamata & Hirata, 1999; CitationGenestra, 2007). This hypothesis is also supported by the results observed in the ALC 60 group, which showed higher CAT activity and cell proliferation rate closer to control levels when compared to the results of the ALC 120 and T + VE + ALC 120 groups.
CitationWarnakulasuriya et al. (2008) demonstrated that drinkers who had been diagnosed with oral premalignant lesions or squamous cell carcinoma showed higher lipid peroxidation levels on lesion biopsies. Nevertheless, it is difficult to define if these oxidative stress indicators are the source or the consequence of molecular alterations presented in those lesions. We agree with these authors when they stated that the exact role of alcohol consumption in oral carcinogenesis remains obscure, but our findings so far suggest that it may act as a tumor promoter and that it may lead to cumulative errors by cell cycle acceleration.
Oral administration was the route of choice for vitamin E, because it would be the most appropriate method for administration in human beings. Although some authors consider Tween 80 to be a suitable vehicle for the preparation of vitamin E solution (CitationKrishnamurthy & Bieri, 1963; CitationKalender et al., 2004; CitationOhta et al., 2006) the pro-oxidant effect observed in our previous study demonstrated that this method should be avoided. In future studies, an alternative is the dissolution of vitamin E in another vehicle, such as corn oil (CitationDomitrovic et al., 2008). Another possibility would be to use a lower concentration of Tween in the solution, such as 0.01%, that did not appear to be noxious (CitationCampos et al., 2005). In this study, Tween 80 was chosen because: (i) it allows the preparation of a water-soluble mixture, which was suitable in order to improve gastrointestinal absorption and (ii) it was used in other studies (CitationKrishnamurthy & Bieri, 1963; CitationIlavazhagan et al., 2001) without any adverse effects. Unfortunately, since Tween reduced Nrf2, suggesting a pro-oxidant effect, the antioxidant (CitationKrishnamurthy & Bieri, 1963; CitationIlavazhagan et al., 2001; CitationKalender et al., 2004) and/or antiproliferative (CitationVincon et al., 2003) effects of vitamin E could not be properly evaluated in this study. Furthermore, our findings indicate that signaling for cell proliferation occurs by means of hydrogen peroxide in oral mucosa, which is in accordance with previous studies in other tissues (CitationBurdon, 1995; CitationLi & Spector, 1997).
The regulation of cell proliferation involves a complex network of pathways that establish cross talk with one another. Since our results have not shown an increase in oxidative stress parameters, i.e., TBARS and carbonyl groups in animals treated with alcohol, oxidative stress cannot be implicated in the increase in alcohol-related cell proliferation in the present study. On the other hand, two main points may be supported by the present data: (i) modifications on antioxidant enzymes indicated that alcohol consumption causes an oxidative imbalance; (ii) CAT activity has an indirect association with cell proliferation in tongue tissues. This is probably a consequence of the action of CAT on the substrate, i.e., hydrogen peroxide, which plays a role in cell signaling.
Conclusion
In conclusion, the present findings suggest that the alcohol-related damage in oral mucosa is cumulative and that the mechanisms involved are complex. Short-term exposure to alcohol induces changes in the redox balance (CitationCarrard et al., 2009) and an increase in cell proliferation appears later, possibly as a consequence of longer periods of biochemical imbalance. Based on the data obtained in the present study, we suggest the involvement of hydrogen peroxide on cell proliferation, but not in relation to the increase in alcohol-related epithelial proliferation. Additionally, our findings support that Nrf2 is somehow implicated in that mechanism. In spite of contributing to alcohol-related damage, Nrf2 did not establish cross talk with the mechanism that led to the increase in epithelial proliferation. Therefore, further studies are necessary in order to clarify the mechanisms of alcohol-related damage in oral mucosa and its role in relation to oral cancer.
Acknowledgements
The authors would like to thank Sabrina P. Moure, Fernanda Visioli and Elisabete U. Rojas for assistance in surgical procedures as well as to Caren Bavaresco, Christiane Gerhard, Mailing Leitão, Adriana Aguiar, Leandro Nunes and Luciana Adolfo for excellent technical support. The authors thank also Malvina Vianna da Rosa, FEPPS (Fundação Estadual de Produção e Pesquisa em Saúde) and Scientific Linguagem, Lauren Valentim, Rafael Dupont and Lívia Wolffenbüttel for the English revision.
Declaration of interest
This study was conducted with the support of a grant received from CAPES (Brazilian Agency for the Improvement of Higher Education Personnel) and CNPq (National Council for Scientific and Technological Development).
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