Role of Hydrogen Sulfide in Oral Disease.

Oral diseases are among the most common human diseases yet less studied. These diseases affect both the physical, mental, and social health of the patients resulting in poor quality of life. They affect all ages, although severe stages are mostly observed in older individuals. Poor oral hygiene, genetics, and environmental factors contribute enormously to the development and progression of these diseases. Although there are available treatment options for these diseases, the recurrence of the diseases hinders their efficiency. Oral volatile sulfur compounds (VSCs) are highly produced in oral cavity as a result of bacteria activities. Together with bacteria components such as lipopolysaccharides, VSCs participate in the progression of oral diseases by regulating cellular activities and interfering with the immune response. Hydrogen sulfide (H2S) is a gaseous neurotransmitter primarily produced endogenously and is involved in the regulation of cellular activities. The gas is also among the VSCs produced by oral bacteria. In numerous diseases, H2S have been reported to have dual effects depending on the cell, concentration, and donor used. In oral diseases, high production and subsequent utilization of this gas have been reported. Also, this high production is associated with the progression of oral diseases. In this review, we will discuss the production of H2S in oral cavity, its interaction with cellular activities, and most importantly its role in oral diseases.

Oral peroxidases: From antimicrobial agents to ecological actors (Review).

Sialoperoxidase and myeloperoxidase are the two main peroxidase enzymes found in the oral cavity. Sialoperoxidase is present in salivary secretions and in the biofilms that line the oral surfaces, while myeloperoxidase is abundant in the dento­gingival sulcus area. In the presence of hydrogen peroxide (H2O2), oral peroxidases catalyze the oxidation of the pseudohalide anion thiocyanate (SCN­) to hypothiocyanite (OSCN­), a strong oxidant that serves an antimicrobial role. Furthermore, oral peroxidases consume bacteria­produced H2O2 and could help inactivate toxic carcinogenic and genotoxic substances. Numerous in vitro studies have reported the antibacterial, antimycotic and antiviral role of peroxidases, suggesting possible applications in oral therapy. However, the use of oral hygiene products incorporating peroxidase systems has not yet been shown to be beneficial for the treatment or prevention of oral infections. This paradox reflects our incomplete knowledge of the physiological role of peroxidases in a complex environment, such as the oral region. While hygiene is crucial for restoring oral microbiota to a symbiotic state, there are no data to suggest that the addition of a peroxidase per se can create a dysbiotic state. Recent investigations have associated the presence of peroxidase activity with gram­positive cocci microbial flora, and its insufficiency with dysbiosis has been linked to pathologies, such as caries, periodontitis or infections of the oral mucosa. Therefore, oxidants generated by oral peroxidases appear to be an essential ecological determinant for oral health through the selection of a symbiotic microbiota capable of resisting oxidative stress. The objective of the present review was to update the current knowledge of the physiological aspects and applications of oral peroxidases in clinical practice.

Salivary Biomarkers and Their Application in the Diagnosis and Monitoring of the Most Common Oral Pathologies.

Saliva is a highly versatile biological fluid that is easy to gather in a non-invasive manner-and the results of its analysis complement clinical and histopathological findings in the diagnosis of multiple diseases. The objective of this review was to offer an update on the contribution of salivary biomarkers to the diagnosis and prognosis of diseases of the oral cavity, including oral lichen planus, periodontitis, Sjögren's syndrome, oral leukoplakia, peri-implantitis, and medication-related osteonecrosis of the jaw. Salivary biomarkers such as interleukins, growth factors, enzymes, and other biomolecules have proven useful in the diagnosis and follow-up of these diseases, facilitating the early evaluation of malignization risk and the monitoring of disease progression and response to treatment. However, further studies are required to identify new biomarkers and verify their reported role in the diagnosis and/or prognosis of oral diseases.

[Physiological and pathological effects of nitric oxide and nitric oxide synthase in oral cavity].

Nitric oxide (NO) is a short-lived free radical which is not only involved in regulating many physiological processes of the body, but also closely related to many diseases. Nitric oxide synthase (NOS) is the key enzyme for NO production. NOS exists as three distinct isoforms, the endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). It has been found that nNOS and eNOS were expressed in normal pulp tissues, periodontal tissues and salivary glands, and the NO produced from nNOS and eNOS was involved in their physiological functions. NO and iNOS are involved in the occurrence and development of pulpitis, periodontitis, salivary gland disease and oral cancer. This review focuses on the physiological and pathological effects of NO and different subtypes of NOS in oral cavity.

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

Acid induced reduction of the glycaemic response to starch-rich foods: the salivary α-amylase inhibition hypothesis.

Numerous studies have reported that the glycaemic response to starch-rich meals can be reduced by 20-50% with acidic drinks or foods. A number of candidate explanations have been put forward, but this phenomenon still remains vaguely understood. This study intends to demonstrate the remarkable effect of acid inhibition of salivary α-amylase during oro-gastric hydrolysis of starch, shedding light on this often overlooked mechanism. Oro-gastric digestions of bread, wheat and gluten-free pastas, combined with either water or lemon juice were performed using a dynamic in vitro system that reproduces gastric acidification kinetics observed in humans. In the presence of water, large proportions of starch (25-85%) and oligosaccharides (15-50%) were released from all foods within the first hour of gastric digestion (pH > 3.5). In the presence of lemon juice (pH < 3.5 at all time), starch release was about twice as low, and amylolysis into oligosaccharides was completely interrupted. Acid-inhibition of salivary α-amylase may explain, at least in part, the reduction of the blood glucose response through acidification of starch-rich foods/meals. This offers new perspectives for the development of strategies to improve the glycaemic response elicited by starch-rich diets.

Chewing bread: impact on alpha-amylase secretion and oral digestion.

During chewing, saliva helps in preparing the food bolus by agglomerating the formed particles, and it initiates enzymatic food breakdown. However, limited information is actually available on the adaptation of saliva composition during the oral processing of complex foods, especially for foods that are sensitive to salivary enzymes. We addressed this question in the context of starch-based products and salivary alpha-amylase. The objectives were two-fold: (1) to determine if salivary alpha-amylase secretion can be modulated by the bread type and (2) to evaluate the contribution of the oral phase in bread enzymatic breakdown. Mouthfuls of three different wheat breads (industrial, artisan and whole-meal breads) were chewed by twelve subjects. Saliva samples were collected at rest and at different times corresponding to 33, 66 and 100% of the individual's chewing sequence. Alpha-amylase activity and total protein content were determined for all saliva samples that were collected. Additionally, the salivary maltose concentration was measured as a marker of bread enzymatic digestion. Boluses were collected at the swallowing time to evaluate the saliva uptake. Chewing industrial bread induced higher saliva uptake than the other breads despite a similar chewing duration. The evolution of salivary amylase activity tended to depend on the type of bread and was highly influenced by a large degree of inter- and intra-subject variability. The protein and maltose concentration steadily increased during chewing as a result of bread breakdown. The salivary protein concentration was mainly affected by the release of the water-soluble proteins of the bread. The salivary maltose concentration was found to be significantly lower for the whole-meal bread. When considering the weight of the mouthful, enzymatic breakdown was found to be most efficient for the breads ranking from industrial > artisan > whole-meal.

Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity.

Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins are the primary carbon sources; however, simple sugar availabilities are limited due to low diffusion from saliva to biofilm. We hypothesized that bacterial glycoside hydrolase (GH) activities would be higher in a biofilm than in saliva in order to maintain metabolism in a low-sugar, high-glycoprotein environment. Salivary bacteria from 13 healthy individuals were used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting carbon sources to a complex carbohydrate. All six GHs measured were higher in vitro when grown in the medium with complex carbohydrate as the sole carbon source. We then collected saliva and overnight dental plaque samples from the same individuals and measured ex vivo activities for the same six enzymes to determine how oral microbial utilization of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in overnight dental plaque. Overall higher GH activities were observed in plaque samples, in agreement with in vitro observation. A similar pattern was observed in GH activity profiles between in vitro and ex vivo data. 16S rRNA gene analysis showed that plaque samples had a higher abundance of microorganisms with larger number of GH gene sequences. These results suggest differences in sugar catabolism between the oral bacteria located in the biofilm and those in saliva.

[A study of urease activity of mouth cavity microflora].

STUDY OBJECTIVE: Analysis of the prevalence of mouth cavity urealytic microflora and determination of the level of its enzymatic activity depending on concentration and amount of urea solution taken as a substrate. MATERIALS AND METHODS: 62 randomly chosen patients at the age of 5-64 took part in the study. Each of them rinsed the mouth with 50 ml of 1% urea solution. Before and after rinsing the concentration of ammonia in the mouth cavity air was measured. In patients with highest and lowest activity of mouth cavity urealytic microflora a series of tests was carried out including mouth rinsing with urea solution in various concentrations and amounts and measuring ammonia concentration before and after rinsing. Obtained results were analyzed using mathematical statistics methods. RESULTS: It was found that in 91% ± 1.8% of randomly chosen patients (p < 0.05) mouth cavity microflora showed apparent urease activity. The lowest concentration (0.0625% in 50 ml) and volume (0.5 ml of 1% solution) levels of urea solution were obtained that can exert negative influence on the results of helicobacteriosis diagnosis by means of mouth cavity air analysis. CONCLUSION: Urealytic microflora in the mouth cavity is very common and may constitute a factor that decreases the specificity of helicobacteriosis diagnosis by means of the methods based on detection of indicators of gas metabolites resulting from the enzymatic reaction in air samples taken from the mouth cavity after oral administration of urea.

The influence of monoamine oxidase variants on the risk of betel quid-associated oral and pharyngeal cancer.

Betel quid (BQ) and areca nut (AN) (major BQ ingredient) are group I human carcinogens illustrated by International Agency for Research on Cancer and are closely associated with an elevated risk of oral potentially malignant disorders (OPMDs) and cancers of the oral cavity and pharynx. The primary alkaloid of AN, arecoline, can be metabolized via the monoamine oxidase (MAO) gene by inducing reactive oxygen species (ROS). The aim of this study was to investigate whether the variants of the susceptible candidate MAO genes are associated with OPMDs and oral and pharyngeal cancer. A significant trend of MAO-A mRNA expression was found in in vitro studies. Using paired human tissues, we confirmed the significantly decreased expression of MAO-A and MAO-B in cancerous tissues when compared with adjacent noncancerous tissues. Moreover, we determined that MAO-A single nucleotide polymorphism variants are significantly linked with oral and pharyngeal cancer patients in comparison to OPMDs patients [rs5953210 risk G-allele, odds ratio = 1.76; 95% confidence interval = 1.02-3.01]. In conclusion, we suggested that susceptible MAO family variants associated with oral and pharyngeal cancer may be implicated in the modulation of MAO gene activity associated with ROS.

Pepsin and amylase in oral and tracheal secretions: a pilot study.

BACKGROUND: Endotracheal intubation increases the risk for microaspiration of secretions around the tube cuff. Pepsin has been used as a biomarker for gastric aspiration. Amylase is a newer proposed biomarker for aspiration of oral contents. OBJECTIVE: To assess the presence of pepsin and amylase in paired oral-tracheal secretions of adult patients treated with mechanical ventilation. METHODS: In this descriptive study, paired samples of oral and tracheal secretions were obtained from adult patients at baseline and again within 4 hours when a need for endotracheal suctioning was assessed. Assays of pepsin and amylase were processed in a specialty diagnostic laboratory. RESULTS: The sample consisted of 10 men and 3 women with a median age of 56 years. The majority were intubated with a subglottic suction endotracheal tube (9 patients, 69%), receiving synchronized intermittent mandatory ventilation (10 patients; 77%), and receiving enteral feedings (11 patients; 85%) through a tube distally placed in the stomach (8 patients; 67%). Pepsin was present in oral secretions of 9 patients (69%), and in tracheal specimens of 7 patients (54%) at one or both sampling times. Amylase was detected in all patients' oral secretions and in tracheal secretions of 5 patients (38%) at one or both sampling times. CONCLUSIONS: Many patients had pepsin, amylase, or both in tracheal aspirates. Pepsin was more commonly detected than was amylase. Although the relationship of this finding to long-term outcomes was not assessed, findings indicate that microaspiration of oral and gastric secretions occurs frequently.

Niche-specific requirement for hyphal wall protein 1 in virulence of Candida albicans.

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.

Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake. METHODS: Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit. Sample suitability was assessed and quality control analyses were conducted. RESULTS: Salivary SLPI was successfully recovered from oral gargles with low intra-assay and high inter-individual variability. Initial measurements showed that salivary SLPI varied considerably across individuals, and that SLPI was inversely associated with age. CONCLUSIONS: This optimized assay can be used to examine the role of SLPI in the acquisition of oral HPV and other infections.

Areca nut extract induces pyknotic necrosis in serum-starved oral cells via increasing reactive oxygen species and inhibiting GSK3ß: an implication for cytopathic effects in betel quid chewers.

Areca nut has been proven to be correlated with various pathologic alterations in oral cavity. However, the mechanisms for such cytopathic effects are still elusive due mostly to the limitations of cell culture systems. Here we discovered that areca nut extract (ANE) induced production of autophagosome vacuoles in cells cultured with rich medium but induced pyknosis and ballooning, two morphological alterations frequently observed in betel quid chewers, in cells under a serum-free culture condition. Permeability of the serum-starved cells to propidium iodide (PI) confirmed ANE induced novel necrosis with pyknosis (pyknotic necrosis), providing a possible explanation for inflammatory infiltration in chewers' mucosa. In these serum-starved cells, ANE strongly induced reactive oxygen species (ROS), which acted as a key switch for the initiation of pyknotic necrosis. Calcium flux was also involved in the morphological alterations. Besides, inhibition of GSK3ß by SB216763 significantly exacerbated the pyknotic necrosis either induced by ANE or H2O2 in serum-starved cells, suggesting that GSK3ß is a critical regulator for ANE/ROS-mediated pyknotic necrosis. Interestingly, LC3-II transition and PARP cleavage were still detected in the serum-starved cells after ANE treatment, suggesting concurrent activation of apoptotic and autophagic pathways. Finally, insulin could counteract the effect of ANE-induced pyknotic necrosis. Taken together, these data provide a platform for studying ANE-induced cytopathogenesis and the first clinical implication for several pathological alterations, such as ballooning and inflammatory infiltration, in betel quid chewers.

Fluctuating roles of matrix metalloproteinase-9 in oral squamous cell carcinoma.

One hallmark of cancer is the degradation of the extracellular matrix (ECM), which is caused by proteinases. In oral cancers, matrix metalloproteinases (MMPs), especially MMP-9, are associated with this degradation. MMPs break down the ECM allowing cancer to spread; they also release various factors from their cryptic sites, including cytokines. These factors modulate cell behavior and enhance cancer progression by regulating angiogenesis, migration, proliferation, and invasion. The development of early metastases is typical for oral cancer, and increased MMP-9 expression is associated with a poor disease prognosis. However, many studies fail to relate MMP-9 expression with metastasis formation. Contrary to earlier models, recent studies show that MMP-9 plays a protective role in oral cancers. Therefore, the role of MMP-9 is complicated and may fluctuate throughout the different types and stages of oral cancers.

Detection of pepsin in mouth swab: correlation with clinical gastroesophageal reflux in preterm infants.

OBJECTIVE: To study the relationship between pepsinogen/pepsin in a mouth swab and clinical gastroesophageal reflux (GER) in preterm infants. METHODS: Preterm infants (birth weight ≤ 2000 g) on full enteral feeds were enrolled. Mouth swabs from cheek and below the tongue were collected one, two and three hours after feeding. An enzymatic assay with substrate fluorescein isothiocyanate-casein was used to detect pepsin A and C activities with further confirmation by western blot. Blinded investigators reviewed the infant's medical record to clinically diagnose GER. RESULTS: A total of 101 premature infants were enrolled. Pepsinogen/pepsin was detected in 45/101 (44.5%) infants in at least one sample. A clinical diagnosis of GER was made in 36/101 (35.6%) infants. Mouth swabs were positive in 26/36 (72%) infants with clinical GER and only 19/65 (29%) infants without GER (p < 0.001). Similarly, the levels of pepsinogen/pepsin A and C were higher in the mouth swabs of infants with clinical GER. CONCLUSION: The detection of pepsinogen/pepsin in a mouth swab correlates with clinical GER in premature infants.

Salivary hexosaminidase in smoking alcoholics with bad periodontal and dental states.

BACKGROUND: A sensitive alcohol marker, ß-hexosaminidase (HEX), in the saliva of alcoholics, is investigated for the first time. METHODS: The activity, specific-activity and output of total HEX and its isoenzymes HEX A and HEX B were measured in the saliva of healthy controls (C), alcohol-dependent non-smokers (ANS), and alcohol-dependent smokers (AS). RESULTS: We observed a significantly increased activity/specific-activity and output of HEX A in the ANS and AS groups, due to the inflammatory state of the oral-cavity/salivary-glands. Significantly increased activity of HEX A contributed to an increase in the salivary activity of the total HEX in the ANS group. A significant decrease in the activity/specific-activity of HEX B in AS seemed to be due to HEX B inactivation by cigarette smoke. We noticed a tendency for deteriorated dental state (lower decayed-missing-filled-teeth index - DMFT), worse periodontal state (higher gingival index - GI and papilla-bleeding index - PBI) in AS, and worse periodontal state (higher GI) in ANS, as compared to the controls. We found no differences in the salivary protein concentrations between all groups and decreased salivary flow in both alcoholic groups as compared to the controls. In alcoholics, the area under the curve (AUC) for HEX A activity/specific-activity was significantly greater than for HEX and HEX B. The salivary HEX A activity/specific-activity had good/excellent sensitivity and specificity in smoking and non-smoking alcoholics, whereas salivary HEX and HEX B had poor/fair sensitivity and specificity. CONCLUSIONS: Salivary HEX A may be helpful in the diagnosis of chronic alcohol intoxication, even in smokers.

Overexpression of Rap-1A indicates a poor prognosis for oral cavity squamous cell carcinoma and promotes tumor cell invasion via Aurora-A modulation.

The functions of Rap-1A in oral carcinogenesis are largely unexplored. In this study, we examined the expression of Rap-1A at different malignant stages of oral cavity squamous cell carcinoma (OCSCC). Semiquantitative RT-PCR, quantitative RT-PCR, and Western blotting were used to evaluate Rap-1A mRNA and protein expressions, respectively, in paired OCSCC patient specimens. To determine the possible correlation between Rap-1A expression and various clinical characteristics, 256 samples from patients with OCSCC were evaluated by immunohistochemical staining. Strong Rap-1A expression was a significant prognostic marker and predictor of aggressive OCSCC. The overall and disease-specific 5-year survival rates were significantly correlated with strong expression of Rap-1A (P < 0.001). Functionally, overexpressed Rap-1A could promote oral cancer cell migration and invasion by Transwell chambers and wound healing assay. Conversely, the suppression of Rap-1A expression using Rap-1A-mediated siRNA was sufficient to decrease cell motility. Furthermore, our data also illustrated that Aurora-A could not only induce mRNA and protein expressions of Rap-1A for enhancing cancer cell motility but also co-localize and form a complex with Rap-1A in the oral cancer cell line. Finally, immunohistochemical staining, indirect immunofluorescence, and Western blotting analysis of human aggressive OCSCC specimens revealed a significantly positive correlation between Rap-1A and Aurora-A expression. Taken together, our results suggest that the Aurora-A/Rap-1A pathway is associated with survival, tumor progression, and metastasis of OCSCC patients.

Effect of particle size on solubility, dissolution rate, and oral bioavailability: evaluation using coenzyme Q10 as naked nanocrystals.

In this paper work, four naked nanocrystals (size range 80-700 nm) were prepared without any surfactant or polymer using the solvent/nonsolvent method. The effects of particle size on their solubility, dissolution, and oral bioavailability were investigated. Solubility and dissolution testing were performed in three types of dissolution medium, and the studies demonstrated that the equilibrium solubilities of coenzyme Q10 nanocrystals and bulk drugs were not affected by the dissolution media but the kinetic solubilities were. Kinetic solubility curves and changes in particle size distribution were determined and well explained by the proposed solubilization model for the nanocrystals and bulk drugs. The particle size effect on dissolution was clearly influenced by the diffusion coefficients of the various dissolution media, and the dissolution velocity of coenzyme Q10 increased as particle size decreased. The bioavailability of coenzyme Q10 after oral administration in beagle dogs was improved by reducing the particle size. For 700 nm nanocrystals, the AUC0₋48 was 4.4-fold greater than that for the coarse suspensions, but a further decrease in particle size from 700 nm to 120 nm did not contribute to improvement in bioavailability until the particle size was reduced to 80 nm, when bioavailability was increased by 7.3-fold.

The effect of chronic alcohol intoxication and smoking on the activity of oral peroxidase.

Peroxidase is the most important antioxidant enzyme in saliva. Through peroxidation of thiocyanate in the presence of H2O2, peroxidase catalyses the formation of bacteriocidic compounds such as hypothiocyanate.The purpose of this study was to evaluate the effect of chronic alcohol intoxication and smoking on the activity of oral peroxidase (OPO). A total of 37 volunteers participated in the study. This cohort consisted of 17 male alcohol-dependent smoking patients after chronic alcohol intoxication (AS group, alcohol + smoking) (mean age: 42 years; range: 26-55) (100-700 g/day of alcohol; 10-20 cigarettes/day) and 20 control male social drinkers(CNS group, control non-smokers) with no history of alcohol abuse or smoking (mean age: 42 years; range:30-53). Salivary peroxidase activity was measured by the colorimetric method. The differences between groups were evaluated using the Mann-Whitney U test. There was significantly higher activity of OPO (p = 0.00001)and significantly lower salivary flow (SF) (p = 0.007) in alcohol-dependent smokers after chronic alcohol intoxication compared to the control group. OPO activity significantly correlated with the number of days of alcohol intoxication, but not with smoking. Gingival index (GI) was significantly higher in smoking alcohol-dependent persons than in the control group, and correlated with OPO activity. The sensitivity of the OPO test was 70% in smoking alcoholics, while specificity was 95%. The increased activity of OPO suggests chronic oxidative stress is more likely due to ethanol action than to smoking. Smoking alcohol-dependent persons have a worse periodontal status than controls. OPO activity as a marker of chronic alcohol abuse may help in the diagnosis of alcoholism.