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. 2020 Jul 30;15(7):e0236161. doi: 10.1371/journal.pone.0236161

Ethanol binge drinking exposure affects alveolar bone quality and aggravates bone loss in experimentally-induced periodontitis

Deborah Ribeiro Frazão 1,#, Cristiane do Socorro Ferraz Maia 2,#, Victória dos Santos Chemelo 1, Deiweson Monteiro 1, Railson de Oliveira Ferreira 1, Leonardo Oliveira Bittencourt 1, Gabriela de Souza Balbinot 3, Fabrício Mezzomo Collares 3, Cassiano Kuchenbecker Rösing 4, Manoela Domingues Martins 5, Rafael Rodrigues Lima 1,*
Editor: Timothy Damron6
PMCID: PMC7392256  PMID: 32730269

Abstract

Background

Periodontitis is a multifactorial inflammatory disease of tooth supporting tissues caused by oral biofilms, influenced by environmental and genetic factors, among others. Ethanol consumption has been considered a factor that enhances alveolar bone loss, especially in high doses. The present study aims to investigate the changes promoted by ethanol binge drinking per se or associated with ligature-induced periodontal breakdown on alveolar bone loss.

Materials and methods

Thirty-two Wistar rats were randomly allocated into four groups: control (C), ethanol (3g/kg/day; 3 days On-4 days Off protocol by gavage for 28 days, EtOH), experimental periodontitis (EP) and experimental periodontitis plus ethanol administration (EP+EtOH). On day 14th, periodontitis was induced by ligatures that were placed around the lower first molars. On day 28th, the animals were euthanized and mandibles were submitted to stereomicroscopy for exposed root area analysis and micro-computed tomography (micro-CT) for the evaluation of alveolar bone loss and microstructural parameters.

Results

The results revealed that ligature-induced alveolar bone loss is aggravated by ethanol binge drinking compared to controls (1.06 ± 0.10 vs 0.77 ± 0.04; p<0.0001). In addition, binge drinking per se altered the alveolar bone quality and density demonstrating a reduction in trabecular thickness, trabecular number parameter and bone density percentual. Periodontal disorder plus ethanol binge drinking group also demonstrated reduction of the quality of bone measured by trabecular thickness.

Conclusions

In conclusion, intense and episodic ethanol intake decreased alveolar bone quality in all microstructural parameters analyzed which may be considered a modifying factor of periodontitis, intensifying the already installed disease.

Introduction

Periodontitis is a multifactorial inflammatory disease, which affects the tooth supporting tissues (i.e., periodontal ligament, cementum, and alveolar bone). Periodontal destruction has been observed among 60–70% of the global population. As a multifactorial disease, 20% of the clinical findings of periodontitis are related to oral biofilms, however, the other 80% are linked to behavioral, environmental and genetic factors, which can modify periodontitis progression. In addition, immunological host response plays a pivotal role in prognosis of periodontally diseased individuals [1, 2].

Some medical conditions, smoking, and social history are described as disease modifiers. They seem to act altering the expression of periodontal disease. However, the exact role of systemic diseases and exposure to different risk indicators in initiating or modifying the progress of the periodontal disease is complex and not fully understood. Diabetes and smoking are the know true risk factors for periodontal disease. Diabetes is characterized as a progressive factor if the glycemic levels are above ideal [3].

Another factor, classified as behavioral, is smoking, which is associated with increased alveolar bone loss. As smoking is often related to alcohol consumption, the toxicological effects caused by ethanol have been related as possible alveolar bone loss intensified by its consumption [4]. Limited studies have linked alcohol consumption and periodontitis [5–7], however, evidence suggests that the relationship exists with several gaps on the exact interaction between alcohol and periodontitis [5, 8]. In the relationship between alcohol exposure and occurrence and progression of periodontitis, a J-curve has been proposed, in which low doses tend to be associated with lower progression and high doses with a more aggressive pattern of progression. Our group has been working on the association between alcohol intake and oral health consequences in animal models both on a heavy alcohol exposure paradigm [9, 10] and with binge drinking-type protocol [11].

Ethanol harmful effects in the oral cavity seem to be proportional to the alcohol-exposure pattern [7, 12, 13]. Enzymatic induction, immune modifier, as well as cementum formation and osteoblast activity reduction have been suggested to alcohol-related oral cavity health disorders [8, 10].

In addition, binge drinking pattern has been reported as the principal type of ethanol exposure among adolescents and adults [14]. Such “social behavior”, characterized by large amounts of alcohol in a short period of time, provokes overspread damages in several organs and tissues, and is not restricted to the central nervous system or liver [15]. The exact pathogenic mechanism that underlies ethanol harmful effects has been extensively investigated, however, it has been postulated that inflammatory and oxidative damage are two important pathways on the alcohol-related tissue damage [16]. Both ethanol pathological via that was described above are shared by periodontitis [17]. The aim of the present study was to assess the effect of binge drinking on alveolar bone loss in Wistar rats. We hypothesize that prolonged binge drinking exposure is associated with the development of spontaneous periodontitis. Our second hypothesis relies on ethanol exposure increasing the impairment caused by ligature-induced periodontitis.

Material and methods

Animals

Wistar rats (90 days-old; n = 32), obtained from animal facility (Federal University of Pará), were randomly allocated to cages (n = 4), with controlled food (NUVITAL®, 3 pellets/animal) and water ad libitum. A 12 h light/dark cycle (lights on 7 AM) and temperature control (25±1°C) was used. All procedures were approved by the Ethics Committee on Experimental Animals of the UFPA (under number 6896071217), according to NIH Guide for the Care and Use of Laboratory Animals recommendations.

Experimental procedures

Fig 1 describes the study protocol. Animals were divided into 4 groups. In the control group (C, G1), animals received distilled water. Ethanol group (EtOH, G2) received ethanol binge drinking protocol characterized by 30% w/v ingestion in 4 sections of 3 consecutive days On-4 days Off protocol by gavage [18]. On the 14th day of experiment, the animals from experimental periodontitis group (EP, G3) and experimental periodontitis + ethanol group (EP + EtOH, G4) were submitted to intraperitoneal anesthesia [xylazine 2% (2mg/ml) plus ketamine 10% (10mg/ml)] and the ligature-induced periodontitis was installed by insertion of cotton ligatures (Coats Corrente, São Paulo, SP, Brazil) around the cervical regions of the first inferior molars, which was maintained until euthanasia, as previously described [19]. Ligatures were maintained for 14 days [19, 20]. Following the ethanol binge drinking protocol, animals were euthanized by intraperitoneal anesthesia [xylazine 2% (2mg/ml) plus ketamine 10% (10mg/ml)] followed by cervical dislocation, and jaws were collected. The right hemimandibules were maintained in physiologic serum (NaCl 0.9%) under the refrigerator for stereomicroscopic analyses. The left hemimandibules were maintained in formol solution 4% for micro-computed tomography (micro-CT) evaluation.

Fig 1. Sample description and experimental steps.

Fig 1

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Stereomicroscopic analyses

The right hemimandibles from each animal (n = 8, each group) were examined by a stereomicroscope (Discovery V8 Zeiss, Germany). The samples were immersed in 10% sodium hypochlorite (NaOCl) for three hours and then they were washed in an ultrasonic bath with distilled water for 2 minutes. To make a better differentiation of the cementum-enamel junction (CEJ), the hemimandibles were immersed in 1% methylene blue for 60 seconds. After they dry at room temperature, the samples were fixed with wax n° 9 (Lysanda, Vila Prudente, São Paulo, Brazil) on individual glass slides with the lingual face of the teeth perpendicular to the axis of observation. The pictures were obtained with a 6.1-megapixel camera (Cannon, Powershot A640) coupled with the stereomicroscope (30.0X). Then, the measurement of the exposed root area (ERA) was made through these images, captured with an adequate scale, from the lingual face of the first molar.

Bodyweight evaluation

For bodyweight measurement, all animals were weighted weekly.

Micro-computed tomography (micro-CT) analysis

Animals left hemimandibules were submitted to micro-CT (MicroCT.SMX-90 CT; Shimadzu Corp., Kyoto, Japan). Images were captured under a rotation of 360°, with an intensity of 70kV and 100 mA. After this, images were reconstituted by inspeXio SMX-90CT software (Shimadzu Corp., Kyoto, Japan), with a voxel size of 10 μm and resolution of 1024x1024 which resulted in 541 images per sample. Bone loss was evaluated in height by RadiAnt DICOM Viewer 5.0.1 (Medixant, Poznan, Poland) where the 3D reconstruction of the hemimandibulas was performed. The tridimensional models were placed on a standard position (Fig 1), where the vestibular and lingual tooth face could be observed. Thus, the vertical bone loss was detected through the measurement of the distance between the cementum-enamel junction and the alveolar bone crest at six points of the first inferior molar, (i.e., mesio-buccal, buccal, distobuccal, disto-lingual, lingual and mesio-lingual) [21], performing the average of these regions.

To verify the alveolar bone tissue quality, the ImageJ® (National Institutes of Health, Bethesda, MD, USA) software was used on a set of 70 images from the inferior first molar alveolar bone region. The interradicular region, close to the furcation area, from the inferior first molar, including the cervical third until the middle third of the root (average area of 0.160mm2), was chosen as the standardized region of interest (Fig 1).

The threshold (0–70) was applied to the segmentation of the different scores of gray color present in the image. The plug-in BoneJ (National Institutes of Health, Bethesda, MD, USA) was used to evaluated the trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and the percentual of bone density (%BV/TV).

Statistical analyses

Animals number samples per group were obtained by G*Power software (Statistical Power Analyses 3.1.9.2). Data distribution normality was analyzed by Shapiro-Wilk test. Statistical analyses were performed by one-way ANOVA followed by Tukey test. Bodyweight data was evaluated by repeated measure two-way ANOVA. Student t test was performed to compare two specific groups. All results were expressed as mean ± standard derivation (SD). Statistical differences were adopted when p<0.05. GraphPad Prism 6.0. software (GraphPad, San Diego, CA, USA) was employed for statistical analyses.

Results

Bodyweight evaluation

Mean body weight did not reveal statistically significant differences among groups. (control: 246.1 ± 6.29; ethanol: 230.6 ± 3.45; ligature-induced periodontitis: 238 ± 1.98; ligature-induced periodontitis + ethanol: 254.8 ± 3.87; p = 0.97).

Stereomicroscopic analysis

Exposed root area (ERA)

The stereomicroscopic analyses showed that ethanol binge drinking exposure did not increase the ERA compared to animals that were not exposed to ethanol (2.05 ± 0.08 vs 1.94 ± 0.08; p = 0.90). However, the animals from the EP + EtOH group presented a higher area of exposed root in comparation to ethanol per se protocol (2.88 ± 0.21 vs 2.05 ± 0.08; p = 0.0005) and controls (2.88 ± 0.21 vs 1.94 ± 0.08; p = 0.0002). The graphic representation of the stereomicroscopic analysis is shown on Fig 2.

Fig 2. Exposed root area in mm2 in all experimental groups.

Fig 2

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(A) Results are expressed as mean ± standard error of the number of exposed root area. On the right, representative photomicrographs of hemi-mandibles of the (C) Control group; (EtOH) Ethanol binge drinking group; (EP) Experimental periodontitis group and (EP + EtOH) Experimental periodontitis and Ethanol group. EP+EtOH group presented the highest ERA identified among the groups. Same letters indicate no significant differences between groups (P < 0.05, ANOVA followed by Tukey post hoc test). Scale bar: 0.5mm.

Micro-computed tomography (micro-CT) analysis

Alveolar bone loss

3D hemimandible analyses show that four ethanol binge drinking exposure did not display additional alveolar bone loss as compared to animals that were not exposed to ethanol protocol (0.82 ± 0.08mm vs 0.77 ± 0.04mm; p = 0.49). However, animals submitted to binge drinking protocol plus experimental periodontitis presented an increase in alveolar bone loss compared to ethanol per se protocol (0.82 ± 0.08mm vs 1.06 ± 0.10mm; p = 0.0001), as well as experimental periodontitis (1.06 ± 0.10mm vs 0.92 ± 0.03mm; p = 0.003) and control (1.06 ± 0.10mm vs 0.77 ± 0.04mm; p<0.0001) (Fig 3).

Fig 3. Cementum-enamel junction to alveolar bone crest (CEJ-ABC) distance (mm) in all experimental groups.

Fig 3

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(A) Results are expressed as mean ± standard error of the distance of cementum-enamel junction to alveolar bone crest in mm. On the right panel, there is the 3D reconstruction of hemi-mandibles of the (C) Control group; (EtOH) Ethanol binge drinking group; (EP) Experimental periodontitis group and (EP + EtOH) Experimental periodontitis and Ethanol group. EP+EtOH group presented the highest alveolar bone loss identified by the greatest measure of the ACJ-ABC distance. Same letters indicate no significant differences between groups (P < 0.05, ANOVA followed by Tukey post hoc test).

Alveolar bone quality and bone density

Control group (C) presented the higher trabecular thickness parameter (Tb.Th) values (0.15 ± 0.02mm). Interestingly, four binge drinking exposure in ethanol group showed reduction in Tb.Th even in the absence of experimental-induced periodontitis (p = 0.0468). In experimental periodontitis group (EP) as well as in experimental periodontitis + ethanol (EP + EtOH) a decrease in Tb.Th parameter was observed compared to control and ethanol groups (p<0.0001). However, they were similar indicating that ethanol exposure per se promoted Tb.Th reduction and it was not intensified by experimental periodontitis induction.

In addition to Tb.Th, trabecular number parameter (Tb.N) was also evaluated. Similarly to Tb.Th data, Tb.N was reduced in Ethanol group compared to control (p = 0.0041) and when experimental periodontitis was induced a decrease in bone quality was also detected (i.e., ligature-induced periodontitis and ligature-induced periodontitis plus ethanol binge drinking groups; p<0.0001). In fact, no differences were observed between experimental periodontitis versus experimental periodontitis plus ethanol exposure groups (p = 0.8188), which is in accordance with Tb.Th evaluation, that reflects no synergistic pathological effects in this parameter.

Percent bone density (%BV/TV) was reduced solely in the ethanol binge drinking animals when compared to control group (p = 0.0411). Experimental periodontitis per se did not affect bone density. The graphic representation of all quality and density parameters are shown on Fig 4.

Fig 4. Results from the Micro-CT quality analysis.

Fig 4

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(A) Region of interest (ROI) used for evaluation. (B) Trabecular thickness parameter (Tb.Th) in all experimental groups. (C) Trabecular number parameter (Tb.N). For these results, same letters indicate no significant differences between groups (P < 0.05, ANOVA followed by Tukey post hoc test). (D) Bone density (%) parameter (BV/TV) obtained by micro-computed tomography (micro-CT). (*) indicates significant difference between the groups (p < 0.05).

Discussion

The present study aimed to investigate the effects of intermittent and episodic ethanol exposure in mandibular alveolar bone accompanied or not by experimental-induced periodontitis in rats. Our findings showed, for the first time, that intermittent and episodic ethanol exposure per se did not increase alveolar bone loss, however, the bone quality was altered in all parameters evaluated. In addition, experimentally induced periodontitis aggravated the alveolar bone loss and trabecular thickness when ethanol intake was associated.

Different protocols have tested the effect of ethanol exposure on periodontal diseases in animal models with distinct results [6, 7, 10, 13, 22, 23]. The idea behind such studies comes from the demonstrated association between alcohol consumption, especially in high doses, with periodontal diseases in populational studies even in a longitudinal approach [6]. The studies performed in animals help in the understanding of the biological plausibility of the encountered associations in populations. Our group has investigated the effects of ethanol exposure on the stomatognathic system, both in chronic as well as in a binge drinking protocol [9–11]. In alveolar bone, our previous data demonstrated that chronic heavy ethanol exposure (6.5g/kg/day) from adolescence till adulthood led to increased alveolar bone loss, even in the absence of experimentally-induced periodontitis [10]. Contradictorily, other study reported that different ethanol concentrations consumed for 8 weeks did not increase alveolar bone loss, however, in the presence of ligature-induced periodontitis, alveolar bone loss was higher than in the control group [23].

In the present investigation, we verified that intense and sporadic alcohol intake promoted changes in quality and density of alveolar bone alone and in association with ligature-induced periodontitis. The 3 days On- 4 days-Off ethanol (3g/kg/day) protocol has been tested in animal model, the results demonstrated that it is a valid way to simulate binge drinking [24–26]. Binge drinking protocol has been widely used for investigation related to bone tissue alterations provoked by alcohol intake in a rat model. It has been described that alcohol exposure displayed a disordered expression of genes that play a pivotal role in bone remodeling [27]. In another study, bone resorption under orthodontic treatment was intensified by binge pattern exposure [28]. In addition, reduced bone mineral density and changes in the number of bone tissue biomarkers, such as receptor activator of nuclear factor-kappa-Β ligand (RANKL) and osteoprotegerin (OPG), has been attributed to ethanol intake [29]. However, it is not well established the effects of binge drinking on the vertical alveolar bone loss, as well as on the alveolar bone quality in adult rats. Besides, we investigated if ethanol exposure interferes on alveolar bone loss and quality displayed by ligature-induced periodontal disease.

In order to verify the effects of binge drinking on the vertical alveolar bone loss, as well as on the alveolar bone quality micro-CT was employed [30–33]. Micro-CT has become the gold standard for bone tissue morphology and microstructure analysis in small animal models, such as rats and mice, as it enables high-definition three-dimensional reconstruction of samples and evaluate both cortical and trabecular bone [34]. Firstly, our data revealed that 3 days On- 4 days Off ethanol exposure protocol did not increase vertical alveolar bone loss. The previous study of our group demonstrated that daily administered heavy ethanol exposure from adolescence till adulthood increased alveolar bone loss [10]. These findings suggest that the harmful effects of ethanol exposure on alveolar bone loss, as well as other body tissues, depends on the period of life, as well as the duration of exposure and amount of consumption. In addition to binge drinking exposure, on the 14th day of ethanol exposure, we submitted animals from two groups to ligature-induced periodontitis. Animals that received the associated protocol (ligature-induced periodontitis + binge drinking) presented higher alveolar bone loss compared to the others that were submitted to experimental periodontitis and ethanol exposure protocols. These data suggest that prolonged ethanol exposure may aggravate alveolar bone loss on the presence of periodontal disorder. These results could be explained by the fact that alcohol exposure promotes oxidative damage and pro-inflammatory effects affecting bone homeostasis, accelerating bone resorption process as well as inhibiting bone formation [35–37]. Additionally, recent studies have shown that bone density is reduced by heavy ethanol consumption, with osteocalcin decrease and RANK / OPG ratio increase [8, 22, 35].

Although increased alveolar bone loss was not observed in the group that was solely exposed to ethanol, the alveolar bone quality was negatively altered by binge drinking. In fact, trabecular thickness, trabecular number, and percent bone density was affected by ethanol-exposed animals. A recent study reported that trabecular structures (i.e., thickness and number) were reduced in sub-chronic ethanol consumption protocol [38], which supports our findings. Likewise, it is well established that ethanol consumption displays oxidative unbalance and pro-inflammatory processes [39].

Another very interesting finding of our study is the fact that that ethanol exposure associated with experimental periodontitis aggravated alveolar bone loss and also disrupted bone quality indicators. Similar to ethanol consumption, periodontitis is also related to oxidative imbalance [40, 41]. We suggest that the pathological mechanisms shared by both isolated harmful stimuli, such as ethanol exposure and experimental periodontal disease, may provoke negative synergistic effects, intensifying alveolar bone tissue damage. Alcohol has been studied as a modifying factor of periodontal disease. In humans, a systematic review shows that several studies demonstrated an association between ethanol consumption and dependence with periodontitis. Although the heterogeneity between the articles is high, differing in the method of sample selection, alcohol consumption and alcohol use, periodontal indices and statistical analysis, the trend clearly demonstrates a detrimental effect [42]. Regarding the limits in translational researches, our results using pre-clinical animal model corroborate the idea that alcohol could negatively impact the pathogenesis of periodontitis. One possible explanation for that, is that the increase of C-reactive protein levels, as well as decrease of neutrophil chemotaxis and phagocytosis elicited by ethanol exposure may predispose to infection by periodontal bacteria [6]. In addition, TNF-α and IL-6 levels are higher in alcohol users [29], which might be equally in crevicular fluid, being related to the presence of periodontitis [6]. Furthermore, it is noteworthy that alcohol modulates bone cell activities in a dose-dependent manner. Thus, ethanol exposure causes an imbalance of the OPG / RANK / RANKL system, implicating in reduced osteoblast activity and osteoclastogenesis improvement [8, 10, 36], which consists of processes that also occurs during periodontitis development [43].

Conclusion

It may be concluded that the intense and episodic ethanol intake (“binge pattern”) promoted decrease in quality of alveolar bone in all parameters analyzed. In addition, we demonstrated that experimentally-induced periodontitis was aggravated binge drinking-like alcohol exposure, qualitatively and quantitatively. Thus, alcohol exposure is possibly related to the modification of the progression periodontitis, intensifying bone tissue damage.

Supporting information

S1 File

(DOCX)

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Acknowledgments

The authors are grateful to the Brazilian National Council for Scientific and Technological Development (CNPq) and Programa Nacional de Cooperação Acadêmica na Amazônia—PROCAD/Amazônia da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was partially supported by a grant provided by CNPq - Brazilian National Council for Scientific and Technological Development - 435093/2018-5 (DF, VC, RO, DF, LB, RL), CAPES/PROCAD - Higher Education Improvement Coordination - 23038.005350/2018-78 (CM, GB, FC, CR, MD, RL) and (CAPES) – Finance Code 001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The APC was funded by Pró-Reitoria de Pesquisa e Pós-graduação from Federal University of Pará (PROPESP-UFPA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Lang NP, Bartold PM. Periodontal health. J Periodontol. 2018;89: S9–S16. 10.1002/JPER.16-0517 [DOI] [PubMed] [Google Scholar]
  • 2.Papapanou PN, Sanz M, Buduneli N, Dietrich T, Feres M, Fine DH, et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Periodontol. 2018;89: S173–S182. [DOI] [PubMed] [Google Scholar]
  • 3.Albandar JM, Susin C, Hughes FJ. Manifestations of systemic diseases and conditions that affect the periodontal attachment apparatus: Case definitions and diagnostic considerations. J Periodontol. 2018;89: S183–S203. 10.1002/JPER.16-0480 [DOI] [PubMed] [Google Scholar]
  • 4.Dantas AM, Mohn CE, Burdet B, Zubilete MZ, Mandalunis PM, Elverdin JC, et al. Ethanol consumption enhances periodontal inflammatory markers in rats. Arch Oral Biol. 2012;57: 1211–1217. 10.1016/j.archoralbio.2012.02.008 [DOI] [PubMed] [Google Scholar]
  • 5.Wang J, Lv J, Wang W, Jiang X. Alcohol consumption and risk of periodontitis: A meta-analysis. J Clin Periodontol. 2016;43: 572–583. 10.1111/jcpe.12556 [DOI] [PubMed] [Google Scholar]
  • 6.Wagner MC, Haas AN, Oppermann R V., Rosing CK, Albandar JM, Susin C. Effect of Alcohol Consumption on Clinical Attachment Loss Progression in an Urban Population From South Brazil: A 5-Year Longitudinal Study. J Periodontol. 2017;88: 1271–1280. 10.1902/jop.2017.170231 [DOI] [PubMed] [Google Scholar]
  • 7.Liberman DN, Pilau RM, Gaio EJ, Orlandini LF, Rösing CK. Low concentration alcohol intake may inhibit spontaneous alveolar bone loss in Wistar rats. Arch Oral Biol. 2011;56: 109–113. 10.1016/j.archoralbio.2010.09.012 [DOI] [PubMed] [Google Scholar]
  • 8.Corrêa MG, Gomes Campos ML, Marques MR, Ambrosano GMB, Casati MZ, Nociti FH, et al. Alcohol intake may impair bone density and new cementum formation after enamel matrix derivative treatment: Histometric study in rats. J Periodontal Res. 2016;51: 60–69. 10.1111/jre.12279 [DOI] [PubMed] [Google Scholar]
  • 9.Fernandes LMP, Teixeira FB, Junior SMA, Pinheiro J de JV, Maia C do SF, Lima RR. Immunohistochemical changes and atrophy after chronic ethanol intoxication in rat salivary glands. Histol Histopathol. 2015;30: 1069–1078. 10.14670/HH-11-604 [DOI] [PubMed] [Google Scholar]
  • 10.Bannach SV, Teixeira FB, Fernandes LMP, Ferreira RO, da Silva Santana LN, Fontes-Júnior EA, et al. Alveolar bone loss induced by chronic ethanol consumption from adolescence to adulthood in wistar rats. Indian J Exp Biol. 2015;53: 93–97. [PubMed] [Google Scholar]
  • 11.Fagundes NCF, Fernandes LMP, Paraense RSDO, De Farias-Junior PMA, Teixeira FB, Alves SM, et al. Binge Drinking of Ethanol during Adolescence Induces Oxidative Damage and Morphological Changes in Salivary Glands of Female Rats. Oxid Med Cell Longev. 2016;2016 10.1155/2016/7323627 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dal-Fabbro R, Marques-de-Almeida M, Cosme-Silva L, Capalbo LC, Ervolino E, Cintra LTA, et al. Effects of different alcohol concentrations on the development of apical periodontitis in rats. Arch Oral Biol. 2019;108 10.1016/j.archoralbio.2019.104538 [DOI] [PubMed] [Google Scholar]
  • 13.Wagner MC, da Rocha JM, Gaio EJ, Cavagni J, Carrard VC, Rösing CK. Effect of 15% alcohol dependence on alveolar bone loss and TNF-α secretion in wistar rats. Braz Dent J. 2016;27: 135–140. 10.1590/0103-6440201600545 [DOI] [PubMed] [Google Scholar]
  • 14.(CISA) C de ISS e Á. Relatório Global sobre Álcool e Saúde—2018. In: Brazli [Internet]. 2018. http://www.cisa.org.br/artigo/10049/relatorio-global-sobre-alcool-saude-2018.php
  • 15.NIAAA—National Institute on Alcohol Abuse and Alcoholism. Alcohol’s Effects on Health. 2019. https://www.niaaa.nih.gov/
  • 16.Rocco A, Compare D, Angrisani D, Sanduzzi Zamparelli M, Nardone G. Alcoholic disease: Liver and beyond. World J Gastroenterol. 2014;20: 14652–14659. 10.3748/wjg.v20.i40.14652 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hernández-Ríos P, Pussinen PJ, Vernal R, Hernández M. Oxidative stress in the local and systemic events of apical periodontitis. Front Physiol. 2017;8: 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc. 2013;8: 627–637. 10.1038/nprot.2013.032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Vargas-Sanchez PK, Moro MG, dos Santos FA, Anbinder AL, Kreich E, Moraes RM, et al. Agreement, correlation, and kinetics of the alveolar bone-loss measurement methodologies in a ligature-induced periodontitis animal model. J Appl Oral Sci. 2017;25: 490–497. 10.1590/1678-7757-2016-0517 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Cavagni J, Seibel L, Gaio EJ, Rosing CK. Methodological aspects in the study of periodontal breakdown in rats: influence of the presence and time of ligature. Acta Odontol Latinoam. 2017;30: 39–45. [PubMed] [Google Scholar]
  • 21.Virto L, Haugen HJ, Fernández-Mateos P, Cano P, González J, Jiménez-Ortega V, et al. Melatonin expression in periodontitis and obesity: An experimental in-vivo investigation. J Periodontal Res. 2018;53: 825–831. 10.1111/jre.12571 [DOI] [PubMed] [Google Scholar]
  • 22.Bastos MF, Gaag GLD, Romero JR, Gabrili JJM, Marques MR, Duarte PM. Effects of Cachaça, a typical Brazilian alcoholic beverage, on alveolar bone loss and density: A study in peripubertal rats. Arch Oral Biol. 2014;59: 82–91. 10.1016/j.archoralbio.2013.10.002 [DOI] [PubMed] [Google Scholar]
  • 23.de Souza DM, Ricardo LH, Kantoski KZ, da Rocha RF. Influence of alcohol consumption on alveolar bone level associated with ligature-induced periodontitis in rats. Braz Oral Res. 2009;23: 326–332. 10.1590/s1806-83242009000300017 [DOI] [PubMed] [Google Scholar]
  • 24.de Oliveira Ferreira R, de Brito Silva R, Magno MB, Carvalho Almeida APCPS, Fagundes NCF, Maia LC, et al. Does periodontitis represent a risk factor for rheumatoid arthritis? A systematic review and meta-analysis. Ther Adv Musculoskelet Dis. 2019;11: 1759720X1985851. 10.1177/1759720x19858514 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lamarão-Vieira K, Pamplona-Santos D, Nascimento PC, Corrêa MG, Bittencourt LO, Dos Santos SM, et al. Physical Exercise Attenuates Oxidative Stress and Morphofunctional Cerebellar Damages Induced by the Ethanol Binge Drinking Paradigm from Adolescence to Adulthood in Rats. Oxid Med Cell Longev. 2019;2019 10.1155/2019/6802424 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Callaci John J., Hime Ryan, Lauing Kristen, Wezeman Frederick H. and Brownson K. Binge Alcohol-Induced Bone Damage is Accompanied by Differential Expression of Bone Remodeling-Related Genes in Rat Vertebral Bone. Calcif Tissue Int. 2009;84: 474–484. 10.1007/s00223-009-9240-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Callaci JJ, Himes R, Lauing K, Wezeman FH, Brownson K. Binge alcohol-induced bone damage is accompanied by differential expression of bone remodeling-related genes in rat vertebral bone. Calcif Tissue Int. 2009;84: 474–484. 10.1007/s00223-009-9240-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.De Araujo CM, Johann ACBR, Camargo ES, Tanaka OM. The effects of binge-pattern alcohol consumption on orthodontic tooth movement. Dental Press J Orthod. 2014;19: 93–98. 10.1590/2176-9451.19.6.093-098.oar [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mikosch P. Alcohol and bone. Wiener Medizinische Wochenschrift. 2014;164: 15–24. 10.1007/s10354-013-0258-5 [DOI] [PubMed] [Google Scholar]
  • 30.Dai J, Ma Y, Shi M, Cao Z, Zhang Y, Miron RJ. Initial changes in alveolar bone volume for sham-operated and ovariectomized rats in ligature-induced experimental periodontitis. Clin Oral Investig. 2016;20: 581–588. 10.1007/s00784-015-1531-3 [DOI] [PubMed] [Google Scholar]
  • 31.Hassumi JS, Mulinari-Santos G, Fabris AL da S, Jacob RGM, Gonçalves A, Rossi AC, et al. Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis. J Appl Oral Sci. 2018;26: e20170326 10.1590/1678-7757-2017-0326 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lisboa MRP, Gondim D V., Ervolino E, Vale ML, Frota NPR, Nunes NLT, et al. Effects of Electroacupuncture on Experimental Periodontitis in Rats. J Periodontol. 2015;86: 801–811. 10.1902/jop.2015.140630 [DOI] [PubMed] [Google Scholar]
  • 33.Schmidt APG. DIMORFISMO SEXUAL DA CABEÇA DA MANDÍBULA EM MACACOS CAPUCHINOS: CEBUS APELLA—AVALIAÇÃO POR MICROTOMOGRAFIA COMPUTADORIZADA SEXUAL DIMORPHISM OF THE MANDIBULAR CONDYLE ON CAPUCHINS: CEBUS APELLA—EVALUATION BY COMPUTERIZED MICROTOMOGRAPHY Piracicaba D. 2019.
  • 34.Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Müller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 2010;25: 1468–1486. 10.1002/jbmr.141 [DOI] [PubMed] [Google Scholar]
  • 35.Callaci JJ, Juknelis D, Patwardhan A, Sartori M, Frost N, Wezeman FH. The Effects of Binge Alcohol Exposure on Bone Resorption and Biomechanical and Structural Properties are Offset by Concurrent Bisphosphonate Treatment. Alcohol Clin Exp Res. 2004;28: 182–191. 10.1097/01.ALC.0000108661.41560.BF [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Chakkalakal DA. Alcohol-induced bone loss and deficient bone repair. Alcohol Clin Exp Res. 2005;29: 2077–2090. 10.1097/01.alc.0000192039.21305.55 [DOI] [PubMed] [Google Scholar]
  • 37.Fernández-Solà J, Porta AP. New treatment strategies for alcohol-induced heart damage. Int J Mol Sci. 2016;17 10.3390/ijms17101651 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Martiniakova M, Sarocka A, Babosova R, Grosskopf B, Kapusta E, Goc Z, et al. Changes in the microstructure of compact and trabecular bone tissues of mice subchronically exposed to alcohol. J Biol Res. 2018;25: 1–7. 10.1186/s40709-018-0079-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Das SK, Vasudevan DM. Alcohol-induced oxidative stress. Life Sci. 2007;81: 177–187. 10.1016/j.lfs.2007.05.005 [DOI] [PubMed] [Google Scholar]
  • 40.Castro MML, Duarte NN, Nascimento PC, Magno MB, Fagundes NCF, Flores-Mir C, et al. Antioxidants as Adjuvants in Periodontitis Treatment: A Systematic Review and Meta-Analysis. Oxid Med Cell Longev. 2019;2019: 1–24. 10.1155/2019/9187978 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chen M, Cai W, Zhao S, Shi L, Chen Y, Li X, et al. Oxidative stress-related biomarkers in saliva and gingival crevicular fluid associated with chronic periodontitis: A systematic review and meta-analysis. Journal of clinical periodontology. 2019. 10.1111/jcpe.13112 [DOI] [PubMed] [Google Scholar]
  • 42.Amaral C da SF, Vettore MV, Leão A. The relationship of alcohol dependence and alcohol consumption with periodontitis: A systematic review. J Dent. 2009;37: 643–651. 10.1016/j.jdent.2009.04.011 [DOI] [PubMed] [Google Scholar]
  • 43.Dahiya P, Kamal R, Gupta R, Bhardwaj R, Chaudhary K, Kaur S. Reactive oxygen species in periodontitis. J Indian Soc Periodontol. 2013;17: 411–416. 10.4103/0972-124X.118306 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Timothy Damron

21 May 2020

PONE-D-20-02327

Ethanol binge drinking exposure affects alveolar bone quality and aggravates bone loss in experimentally-induced periodontitis

PLOS ONE

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Frazão and colleagues explores the potential role of ethanol consumption on alveolar bone loss. The study focused on the use of a rats to investigate whether ethanol exposure per se was sufficient to induce dental changes. It is an interesting and important question, and the group used a standard, well-accepted method for inducing periodontal tissue breakdown by placing ligatures around the molars. Four experimental groups were described: a “control” group, an ethanol exposure group in which rats are given ethanol for 3 days then off for 4, for a total of 28 days; another group that received ethanol and ligatures, and a fourth group that has ligatures and ethanol exposure as well.

If I understand the experimental groups correctly, those in G1 are not exposed to ethanol (the control group). Animals in G2 are exposed to ethanol (“Ethanol group (EtOH, G2) received ethanol binge drinking protocol characterized by 30% w/v ingestion…”). Animals in the G3 and G4 groups were also exposed to ethanol for 14 days (“On the 14th day of ethanol exposure protocol, animals from experimental periodontitis (G3) group and experimental periodontitis + ethanol (G4) group were submitted to… ligature-induced periodontal disease by insertion of cotton ligatures”). Consequently, there is no group that received ligatures to induce periodontitis) but did not receive ethanol. Therein lies my major concern with this work: without this group, it is impossible to separate the effects of the ligatures from the effects of ethanol exposure. Consequently, the data do not support the conclusion made by the authors, that binge-drinking decreases alveolar bone quality. (I would prefer that a measurable value be substituted since “bone quality” is a vague and non-quantitative descriptor). Other questions and concerns are outlined below, but in its current form the data do not support the conclusions drawn.

Animals in G4, which had both received ligatures and were exposed to ethanol presented a higher area of exposed root in comparation to “ethanol per se protocol and controls”. I don’t understand this result. If I go by the figure, the dark purple bar representing the G4 group is higher than the light purple G1 bar e.g., there is greater root exposure in G4 compared to G1 but that is easily understood by the fact that there is a ligature in the G4 group; it is expected that the ligature causes gingival recession.

The results in Figure 3 are described as follows: “animals submitted to binge drinking protocol plus experimental periodontitis presented an increase in alveolar bone loss compared to ethanol per se protocol.” The appropriate control, however, is the group that had ligatures but no ethanol exposure; this group does not exist, as far as I can tell.

In Figure 4, it appears that G3 and G4 exhibit an equivalent e.g., non-significant difference in BV/TV, trabecular number, and trabecular thickness. If I understand the groups correctly, that means that whether the animal is exposed to ethanol or not, the effect on alveolar bone loss is equivalent. Thus, I cannot see how

Reviewer #2: Dear Author, congratulations on a well designed study. It is well written and well performed. However, I am missing some more parameters from this study. Since you already did microCT, I think it would be benefical for this study to measure the distance between the cemento-enamel junction and the alveolar bone crest (CEJ-AC) at 6 different points (mesio-buccal, buccal, distobuccal, disto-lingual, lingual and mesio-lingual), noy just the four points that you have chosen, particular when you see the large different in alveolar bone promixal and distal. Please see Virto L, , et al. Melatonin expression in periodontitis and obesity: An experimental in-vivo investigation. J Periodont Res. 2018;53:825–831. on how to do it. It does not take long time, and would give you valuable information on the bone loss similar to how you would evaluated with a periodontal probe in the clinic. It would be beneficial for this study to include histology

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PLoS One. 2020 Jul 30;15(7):e0236161. doi: 10.1371/journal.pone.0236161.r002

Author response to Decision Letter 0

3 Jun 2020

Dear Reviewers,

Thank you for your dedication to revise our manuscript. In this document are the answers to the comments made by reviewers 1 and 2. The answers are described in detail and everything that has been changed in the manuscript is described here.

Referee: 1

1. “[…] Four experimental groups were described: a “control” group, an ethanol exposure group in which rats are given ethanol for 3 days then off for 4, for a total of 28 days; another group that received ethanol and ligatures, and a fourth group that has ligatures and ethanol exposure as well. If I understand the experimental groups correctly, those in G1 are not exposed to ethanol (the control group). Animals in G2 are exposed to ethanol (“Ethanol group (EtOH, G2) received ethanol binge drinking protocol characterized by 30% w/v ingestion…”). Animals in the G3 and G4 groups were also exposed to ethanol for 14 days (“On the 14th day of ethanol exposure protocol, animals from experimental periodontitis (G3) group and experimental periodontitis + ethanol (G4) group were submitted to… ligature-induced periodontal disease by insertion of cotton ligatures”). Consequently, there is no group that received ligatures to induce periodontitis) but did not receive ethanol […]”

Answer:

We apologize for the misunderstanding about that information. We noticed that it was not very clear in the text that the Experimental Periodontitis (EP, G3) group of this work was only submitted to ligature, but without receiving ethanol, receiving distilled water instead, as described in Figure 1. We have already modified this information in the manuscript to increase understanding. Group 2 and group 3 were submitted to separate exposures: one just received ethanol (G2) and the other received induction of experimental periodontitis, without ethanol exposure (G3). Thus, G2 and G3 represent the groups that were exposed to the challenges separately, while Group 4 represents the group that received both challenges (ethanol and ligature-experimental periodontitis) simultaneously.

2. “Animals in G4, which had both received ligatures and were exposed to ethanol presented a higher area of exposed root in comparation to “ethanol per se protocol and controls”. I don’t understand this result. If I go by the figure, the dark purple bar representing the G4 group is higher than the light purple G1 bar e.g., there is greater root exposure in G4 compared to G1 but that is easily understood by the fact that there is a ligature in the G4 group; it is expected that the ligature causes gingival recession.”

Answer:

We understand your question and the opportunity to clarify it. When the G4 (EP + EtOH) is compared with G2 (EtOH), we observe a statistically significant difference, that leads us to understand that ethanol per se did not cause an increase in the exposed root area, but when associated with experimental periodontitis, i.e, when there is a synergistic effect of the two exposures, demonstrated by the increased area. This analysis by stereomicroscope is complementary to the evaluation of alveolar bone loss in height by micro-CT, since it shows us not only how much bone was lost in height, but the root area left exposed due to the process of periodontal breakdown.

3. “The results in Figure 3 are described as follows: “animals submitted to binge drinking protocol plus experimental periodontitis presented an increase in alveolar bone loss compared to ethanol per se protocol.” The appropriate control, however, is the group that had ligatures but no ethanol exposure; this group does not exist, as far as I can tell.”

Answer:

In fact, this group exists, it is group 3, which only underwent to ligature-induced periodontitis without exposure to ethanol. As shown in the graph, in figure 3, and in the text, on lines 137-140, there was a significant difference between group 3 and group 4, showing that when associating periodontitis with ethanol, there was a greater bone loss in height compared to only experimental periodontitis induction.

4. “In Figure 4, it appears that G3 and G4 exhibit an equivalent e.g., non-significant difference in BV/TV, trabecular number, and trabecular thickness. If I understand the groups correctly, that means that whether the animal is exposed to ethanol or not, the effect on alveolar bone loss is equivalent. Thus, I cannot see how.”

Answer:

We understand your question. The data shows that the EtOH (G2) group presented a greater loss of trabecular thickness, trabecular number, and BV/TV compared to the control group. However, the groups with experimental periodontitis (G3 and G4) had a much greater loss compared to G1 and G2, but that was not significantly different from each other. This leads us to believe that because of periodontitis, the microstructural parameters were much more affected compared to the damage caused only by ethanol, which can be explained by the increased inflammation in groups with periodontitis.

Referee: 2

1. “Dear Author, congratulations on a well designed study. It is well written and well performed. However, I am missing some more parameters from this study. Since you already did microCT, I think it would be benefical for this study to measure the distance between the cemento-enamel junction and the alveolar bone crest (CEJ-AC) at 6 different points (mesio-buccal, buccal, distobuccal, disto-lingual, lingual and mesio-lingual), noy just the four points that you have chosen, particular when you see the large different in alveolar bone promixal and distal. Please see Virto L, , et al. Melatonin expression in periodontitis and obesity: An experimental in-vivo investigation. J Periodont Res. 2018;53:825–831. on how to do it. It does not take long time, and would give you valuable information on the bone loss similar to how you would evaluated with a periodontal probe in the clinic. It would be beneficial for this study to include histology”

Answer:

Thank you for your contribution and evaluation of the manuscript. We already evaluated the alveolar bone loss in the micro-CT at 6 points, as described in the lines 95-98 of the article. However, to facilitate understanding, the name of the points used for the evaluation was modified according to the suggested reference (mesio-buccal, buccal, distobuccal, disto-lingual, lingual, and mesio-lingual).

Regarding the histological evaluation, for this work (the first with binge drinking paradigm) we tried to make a diagnostic analysis of this additional exposure pattern. We appreciate the suggestion and intend to use it for future work to evaluate the mechanisms of damage to alveolar bone associated with ethanol binge drinking consumption and experimental periodontitis.

Finally, we would like to thank you for the meaningful contributions and acknowledge that they have contributed substantially to improve the impact of our manuscript. We hope that the changes implemented and the answers provided are enough for the recommendation of the publication of our study in Plos One.

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Decision Letter 1

Timothy Damron

1 Jul 2020

Ethanol binge drinking exposure affects alveolar bone quality and aggravates bone loss in experimentally-induced periodontitis

PONE-D-20-02327R1

Dear Dr. Lima,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Timothy Damron

Academic Editor

PLOS ONE

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Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

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Reviewer #2: no more comments need for this round and I hereby accept the manuscript for publication. Thank you for address the comments made

**********

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Reviewer #2: No

Acceptance letter

Timothy Damron

20 Jul 2020

PONE-D-20-02327R1

Ethanol binge drinking exposure affects alveolar bone quality and aggravates bone loss in experimentally-induced periodontitis

Dear Dr. Lima:

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