This study investigates morphological and biochemistry effects of binge ethanol consumption in parotid (PG) and submandibular (SG) salivary glands of rats from adolescence to adulthood. Female Wistar rats (
According to World Health Organization (WHO), individuals over 15 years of age consume on average 6.2 liters of pure alcohol per year, which translates into 13.5 g of pure alcohol a day. America and Europe are regions shown as consuming above average. In 2012, 5.9% of global mortality was associated with alcohol use [
In this context, there are an increased potential and intense consumption of ethanol (EtOH) in women, especially in regions such as the United States [
Heavy alcohol consumption, with high doses of EtOH for a short period of time followed by a period of abstinence, in a binge style, has a high prevalence and increases in frequency throughout adolescence, peaking in young adults and subsequently declining with advancing age [
EtOH consumption has been associated with triggering damage to organs and body tissues, such as the upper gastric tract, skeletal muscles, and salivary glands, which can cause morphological and functional changes [
Salivary glands and their resulting secretion have a huge importance in the maintenance of oral and general homeostasis [
The investigation of changes in salivary glands after alcohol intake may involve analysis of immunohistochemical markers in parenchyma and stroma, including cytokeratin, vimentin, and alpha smooth muscle actin, intended to evaluate morphological alterations [
This study aimed to evaluate the effects caused by episodic and intense intoxication of EtOH in a 3-day/week binge pattern on the parotid and submandibular salivary glands of female rats, during adolescence to young adulthood phase.
Female Wistar rats, 35 days old, were kept in standard conditions of temperature, in a climate-controlled room on a reverse light/dark cycle of 12 hours (lights on 7:00 AM), with food and water
A sample size calculation was performed assuming a normal distribution of the variables tested. A power of 80% and a bilateral alpha level of 5% were assumed with standard deviation of 1.29 (ethanol group) and 0.20 (control group). Standard deviation was determined through a previous study [
All animals received EtOH at a dose of 3 g/kg/day (20% w/v), simulating a pattern of binge consumption previously described [
The total sample was divided into two groups, according to the period of solution administration: G1, with 1 week of 3 days of exposure to ethanol/distilled water; and G2, with 4 weeks of 3 days of exposure to ethanol/distilled water. Each group was composed of a control group, where the animals received distilled water, and an ethanol group, where animals received EtOH. Twelve hours after treatment, animals were divided and submitted to collection of fresh glands or perfusion (
Sample description and experimental steps. Animals and sample description (a); morphometric analysis steps (b); immunohistochemistry analysis steps (c); oxidative stress assays (d).
Blood Alcohol Concentration (BAC) was obtained on postnatal day 37, after 60 minutes of alcohol intake, in the last three days of the binge drinking model exposure. Blood samples (
The animals were anesthetized with a combination of ketamine hydrochloride (90 mg/kg) and xylazine (10 mg/kg). After the absence of corneal and paw withdrawal reflex, surgery was performed to collect the parotid and submandibular salivary glands. The gland of the right side was removed and used for oxidative stress analysis. The gland of the left side was removed after perfusion and used to immunohistochemistry and morphometric analysis.
All glands were weighed after removal by analytical balance (FA 2104 N, Electronic Balance Bioprecisa, Shanghai, China), and the relative glandular weight was calculated (gland weight × 100/final body weight).
After perfusion of the animals, one submandibular and one parotid gland from the left side of each animal were postfixed in 6% formaldehyde until processing [
With regard to morphometric assays, the mean percentage equivalent to the glandular parenchyma and stroma region was evaluated, with evaluation of two slides per animal: one submandibular and one parotid gland. The area of the samples was evaluated by the planimetry method for counting points, using ImageJ software version 1.33-1.34 (NIMH, NIH, Bethesda, MD, USA,
Immunohistochemical studies were performed on paraffin-embedded tissues using the streptavidin (Reveal Spring, Pleasanton, CA, USA) and 3,3-diaminobenzidine (DAB; Sigma, USA) methods. Briefly, 3
Immunohistochemical analysis of two slides per animal was performed, one submandibular and one parotid gland. The cytoplasmatic expression of the proteins studied was measured throughout the glandular parenchyma, by evaluation of the extent of the area (
After collection and weighing of glands, the extracted tissue was rinsed in saline and subjected to freezing in liquid nitrogen and subsequently stored at −80°C. For analysis, samples were thawed and resuspended in 20 mM Tris-HCl buffer, pH 7.4, at 4°C for sonic disintegration (approximate concentration of 1 g/mL) (Figure
The concentration of nitrite was determined based on a reaction with Griess reagent (0.1% naphthyl-ethylene-diamine and 1% sulfanilamide in 5% phosphoric acid; 1 : 1). An aliquot of crude homogenate was centrifuged at 21,000 g for 20 min at 4°C, and the supernatant was used to analyze nitrite levels such as described elsewhere [
Briefly, fifty microliters of the supernatant or standard nitrite solution was added to 50 mL of Griess reagent and incubated for 20 minutes at room temperature. The absorbance was measured at 550
The level of lipid peroxidation was determined by the method proposed by Esterbauer and Cheeseman, based on measurement of malonaldehyde (MDA) and 4-hydroxyalkenals (4-HA) levels [
Quantities of total protein content in the supernatants (used for determination of lipid peroxidation and nitrite levels) were assayed as described previously [
The mean values were obtained from the control and EtOH groups, among parotid and submandibular glands, in each period evaluated, for each morphologic and biochemical assay. The normality of the data was verified by the Shapiro-Wilk test. The Student
The measurement of BAC 1 hour after the last three binge drinking exposures reached
Effects of binge drinking (EtOH administration) during adolescence in the body weight gain of rats (a) and relative gland weight of parotid (b) and submandibular (c) glands. The results were expressed as mean ± SEM after Student’s
In the morphometric assay, as the results show in Figure
Morphometric analysis: effects of binge drinking (EtOH administration) during adolescence and young adulthood. Parotid (a) and submandibular (b) glands, after one week (control: 1, ethanol: 2) and four weeks of ethanol exposure (control: 3, ethanol: 4), according to treatment group. These results were expressed with photomicrographs and mean ± SEM (Mann-Whitney
In parotid glands, ethanol consumption affected expression of CK-18 at one week and four weeks of exposure (Figure
Effects of binge drinking (EtOH administration) during adolescence on the parotid and submandibular gland immune expressions. Anti-
On the other hand, submandibular gland showed no difference between the control and ethanol groups in both periods of analysis regarding the expression of CK-18 (Figure
No difference in nitrites concentration among the control and ethanol groups was detected in parotid glands and submandibular glands (Figures
Effects of binge drinking (EtOH administration) during adolescence and young adulthood on the parotid and submandibular glands. The results were expressed as means ± SEM of the nitrite concentration (pM) per milligram of protein after 1 week (G1) and 4 weeks of ethanol exposure (G2) on parotid (a) and submandibular (c) glands and malondialdehyde (MDA) concentration (in pM) per milligram of protein after 1 week (G1) and 4 weeks of ethanol exposure (G2) on parotid (b) and submandibular (d) glands.
The levels of MDA showed an increase in expression in parotid glands at one and four weeks of exposure to ethanol (Figure
In this study, the effects of an episodic binge drinking model of consumption of ethanol in salivary glands were investigated in female rats considering two different periods, one and four weeks of exposure. Our results showed, for the first time, that a single episode of ethanol binge drinking can affect the parenchyma of the parotid gland, reduce myoepithelial cells, and increase the levels of MDA in the parotid and submandibular glands. Furthermore, a 4-week exposure in the same conditions reveals a reduction in cytokeratin expression and MDA levels of the parotid gland and a reduction of the myoepithelial cells in the submandibular gland.
Our group have studied the effects of alcohol on the salivary glands of female rats from adolescence until adulthood. Our first findings demonstrated that a chronic heavy ethanol paradigm (6.5 g/kg/day) induces morphologic changes in both glands, however, in a different way. In parotid glands, an increase in total gland weight was observed as well as atrophy of glandular parenchyma. On the other hand, heavy ethanol intake promoted an increase in the submandibular gland stroma area. In addition, our work demonstrated through an immunohistochemistry assay that alcohol exposure increased duct-like cells related to caspase-3 overexpression in submandibular glands. These results highlight the difference between parotid glands and submandibular glands in the face of chemical noxious stimuli [
After that, we decided to investigate whether habitual and recreational consumption of alcohol among adolescents promotes the same range of damage in the salivary glands. Therefore, we employed the current protocol in doses that mimic a binge drinking pattern (3 g/kg/day for three days a week) [
The evaluation of two different periods of exposures to EtOH shows the effects of an acute (1 week) and chronic (4 weeks) heavy binge drinking model in salivary glands [
In this study, the ethanol group showed conformity of response with the chronic heavy drinking model in the expression of cytokeratin and
In contrast, neither period of ethanol exposure was able to cause changes in the EtOH group when compared to the control group or modify parotid or submandibular glands in terms of size or parenchyma volume, although it has been shown that heavy chronic consumption of ethanol can increase the size and cause atrophy of the parenchyma area of the parotid gland [
In line with a heavy chronic model of exposure to EtOH, morphologic changes after binge drinking are more related to expression patterns of CK-18 and
Parotid glands showed a reduction in CK-18 expression in 1- and 4-week episodic binges, while submandibular glands did not present any significant modification. A previous study exhibits a reduction of cytokeratin expression in parotid glands and an increase in submandibular glands after chronic ethanol exposure [
The changes observed in
Intended to analyze stromal fibroblasts, vimentin expression was also verified in this study and no difference among groups was detected in either the periods or glands evaluated. Vimentin symbolizes a mesenchymal marker of cell migration and invasion identifying activated fibroblasts or the myofibroblasts in salivary glands [
Alcohol consumption is also connected with a metabolic imbalance of free radical production, leading to oxidative stress. In this study, a higher level of MDA, a lipid peroxidation marker, was demonstrated in the parotid glands of the ethanol group after 1 and 4 weeks of ethanol intake in a binge model. Submandibular glands exhibited a higher level of lipid peroxidation in the ethanol group only after a 1-week binge treatment. No changes between groups were detected in nitric oxide analysis for both glands in 1-week or 4-week binge ethanol exposure. Previously, it was shown that increased levels of lipid peroxidation, detected by the production of toxic compounds such as MDA, were related to membrane damage and can be deleterious for membrane permeability [
A difference in response between parotid and submandibular glands was also shown by Fernandes et al. [
In the present research, differences between parotid and submandibular responses were related. As we know, these glands have metabolic and structural differences. Parotid glands present purely serous acini and submandibular glands show mucous acini carrying a terminal cap of serous cells [
For the first time, in this study, 1 week of episodic binge drinking in female rats has been connected with damage to the submandibular and parotid glands showing a reduction in myoepithelial cells, cytokeratin expression in parenchyma, and higher levels of lipid peroxidation. On the other hand, different patterns of response were observed after 1-week and 4-week exposure to ethanol. Further studies are necessary to clarify the damage with longer periods of exposure to ethanol.
None of the authors have any conflict of interests regarding the publication of this paper.