Research Unit of Alcohol Diseases, Helsinki University Central Hospital, PL 345, Tukholmankatu 8F, 00029 HYKS,
1 Institute of Dentistry, University of Helsinki,
2 Department of Oral and Maxillofacial Surgery, Helsinki University Central Hospital and
3 Anaerobe Reference Laboratory, National Public Health Institute, Helsinki, Finland
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Abstract |
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Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase.
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Introduction |
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In addition to the intracellular formation of acetaldehyde via mucosal alcohol dehydrogenases (ADHs), acetaldehyde is also formed in high concentrations in saliva by the oral microflora (810). Salivary acetaldehyde production shows high interindividual variation (10). Recently, we have been able to demonstrate increased acetaldehyde production in the mouthwashings of patients with upper gastrointestinal tract cancer, but the causal relationships for this finding remain unclear (11).
The aim of the present study was to elucidate the factors that regulate microbial production of acetaldehyde in saliva. Moreover, possible differences in microbial composition and relative concentrations among `high' and `low' acetaldehyde producers were examined.
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Materials and methods |
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Questionnaire
A questionnaire was answered by each volunteer. Information concerning age, gender, tobacco use, alcohol use, diet, oral health status, oral hygiene habits and other characteristics were elicited. Tobacco use indicators included the average number of cigarettes, cigars or pipes smoked per day within the past 30 days, duration of smoking in years and the date when possible smoking cessation occurred. Daily tobacco consumption was calculated as cigarettes smoked per day (1 cigar = 3 cigarettes, 1 pipe = 5 cigarettes). Ex-smokers with smoking cessation of >5 years were ranked in further analyses as non-smokers; ex-smokers with a shorter period of cessation were excluded.
Alcohol consumption was estimated as the average number of ingested drinks (~12 g of pure alcohol) for every drinking day during the past 30 days and as the frequency of alcohol intake per week. Based on these data, the average amount of alcohol consumed as g pure ethanol/day was calculated. Volunteers were ranked as non-drinkers (<1 g/day), moderate drinkers (130 g/day for females, 140 g/day for males) or heavy drinkers (>30 g/day for females, >40 g/day for males). Since the statistical analysis did not reveal significant differences between the teetotallers and moderate drinkers, these groups were combined.
Indicators of oral health included the number of teeth lost (except wisdom teeth), frequency and time since last visit to the dentist, denture wear, frequency of toothbrushing, self-reported periodontitis, self-reported frequency of a dry or burning mouth, frequency of mouthwash use and frequency of eating between meals.
Salivary samples
Stimulated whole saliva was collected between 9 and 12 a.m. after 1 min use of a paraffin chewing gum (Orion Diagnostics, Espoo, Finland). It was immediately frozen at 70°C. Exclusion criteria were as follows: treatment with oral antiseptic or antibiotics in the past month, food or fluid intake, smoking or toothbrushing in the past 90 min, recent alcohol intake or measurable amount of alcohol in the saliva by head space gas chromatography.
Salivary acetaldehyde production capacity
Saliva was thawed and preheated to 37°C before analysis. A sample of 400 µl of saliva was transferred to a gas chromatograph vial. To this was added 50 µl of potassium phosphate buffer (final concentration 100 mM, pH 7.4) containing ethanol (final concentration 22 mM) and the vials were immediately tightly closed. Vials were incubated for 90 min and the reaction was stopped by injecting 50 µl of 6 M perchloric acid through the rubber septum of the vial. All samples were measured as triplicates. For each salivary sample one analysis was carried out by concomitantly adding 50 µl perchloric acid and an ethanol/potassium phosphate buffer mixture, before addition of 400 µl of saliva. These control assays for baseline and artefactual acetaldehyde production were incubated for 90 min and revealed values were subtracted from the acetaldehyde levels after 90 min incubation with ethanol. Head space gas chromatographic conditions (Perkin Elmer, Norwalk, CT) were as follows: column 60/80 Carbopack B/5% Carbowax 20M, 2 mx1/8 inch (Supelco, Bellefonte, PA); oven temperature 85°C; transfer line and detector temperature 200°C; carrier gas flow rate (N2) 20 ml/min.
Bacterial analysis
Among all volunteers, the 10 saliva samples with the lowest and highest acetaldehyde production were identified. The samples were thawed, serially diluted in peptone yeast extract broth and 10 µl of undiluted saliva and the appropriate dilutions were inoculated on several non-selective and selective agar media for the enumeration and isolation of aerobic and anaerobic bacteria. Aerobic blood agar base medium and chocolate agar were used for the determination of total aerobic counts. Vitamin K1- and hemin-supplemented anaerobic Brucella blood agar media were used for the determination of total anaerobic counts. The aerobic plates were incubated at 36°C in an atmosphere containing 5% CO2 for a total of 57 days and anaerobic plates in anaerobic jars filled by the evacuation replacement method with mixed gases (85% N2, 10% CO2, 5% H2) were incubated for 7 days for the first inspection and further up to 14 days for the final inspection. Bacterial counts were determined by multiplying the number of colonies by the dilution factor, adjusted for inoculation volume.
Statistical analysis
For statistical evaluation of the data in Table I, the following statistical calculations were done. As a preliminary analysis, and to test for co-linearity of variables, a Spearman correlation matrix was computed for the entire study population. This was followed by multivariate regression analyses. Variables such as age, smoking (the number of cigarettes smoked per day), alcohol (the average amount of alcohol in grams), tooth brushing, tooth loss (number of lost teeth) and eating between meals were correlated as continuous variables, whereas self-reported periodontitis, frequency of dentist visits, mouthwash use, dentures and self-reported dry mouth and burning mouth were calculated as categorical variables. For categorical values, dummy variables were used in the analysis. As co-linearity was obvious for smoking and heavy alcohol intake, the multivariate analysis were re-run for non-smokers (n = 189) and for moderate and non-drinkers (n = 259, <30/40 g alcohol/day, for females/males, respectively) in order to adjust for this confounding factor. A multiple linear regression analysis, a forward stepwise regression analysis (no variable forced into the equation) and a best subsets regression analysis (r2 as best criterion) was run with the best descriptor for all variables, setting acetaldehyde production as the dependent variable.
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All reported P values derive from two-sided tests. All values are expressed as means ± SEM. For all statistical calculations, statistical software (SigmaStat 2.0; Jandel Scientific, San Rafael, CA) was used.
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Results |
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Age (inverse correlation, r = 0.13, P = 0.02) and the reported frequency of dry mouth (positive correlation, r = 0.1, P = 0.06) were other, less compelling factors possibly associated with increased acetaldehyde production. Again, both factors showed co-linearity with smoking (dry mouth, r = 0.17, P = 0.001; age, r = 0.2, P < 0.0001) and alcohol intake (age, r = 0.32, P < 0.0001). After adjustment for confounders, the reported frequency of a dry mouth showed a slight but significant contribution to salivary acetaldehyde production, at least in non-smokers. It can be estimated that a subject with a very frequent dry mouth has ~20% higher salivary acetaldehyde levels than subjects without such symptoms.
None of the other variables contributed significantly to salivary acetaldehyde production. In detail, patients with cancer of the oral cavity did not have salivary acetaldehyde production which differed significantly from the rest of the cohort. This was true both for patients with fresh, untreated tumours as for patients in follow-up after surgical removal of a malignancy.
Patients were ranked according to their alcohol consumption (moderate/non-drinkers versus heavy drinkers) and smoking habits (non-smoker versus smoker). Both smoking and heavy alcohol consumption independently increased salivary acetaldehyde production in comparison with the control groups by 6075%, and combined misuse further increased it (Figure 1).
Microbial analysis
Microbial analysis of the saliva of `high' and `low' acetaldehyde producers showed a clear trend in aerobic conditions. Total counts of aerobes were significantly increased among `high' producers. Aerobic species that were significantly associated with higher acetaldehyde production were Streptococcus salivarius, hemolytic Streptococcus viridans var., Corynebacterium sp., Stomatococcus sp. and yeasts (Figure 2). Yeasts were not only found at higher concentrations, but also more frequently among the subjects with higher acetaldehyde production (eight of 10 versus two of 10, P = 0.02). Corynebacterium sp. were found in all `high' acetaldehyde producers, but in only six of 10 `low' producers (P = 0.08), whereas the other facultative commensals Streptococcus mutans, Haemophilus sp. and Staphylococcus sp. were equally distributed in both groups. No bacterial species was found to be significantly more frequent in the saliva of `low' acetaldehyde producers (Figure 2
). Although the total anaerobic counts were slightly increased in `high' producers, there was no correlation between single bacterial species and acetaldehyde production (data not shown).
The selection criterion for `high' and `low' acetaldehyde producers was exclusively the measured acetaldehyde level and there was no significant difference in the proportion of smokers (P = 0.19) or heavy drinkers (P = 0.62, calculated by Fisher's exact test) in the two groups.
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Discussion |
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Although alcohol and tobacco smoke are well-known independent and strong risk factors for upper gastrointestinal tract cancer, their combined action on these epithelia is poorly understood. There is epidemiological evidence indicating that alcohol and tobacco act together in a more multiplicative rather than in an additive manner and, accordingly, seem to have synergistic tumour-promoting effects (13,18,19). As alcohol is involved synergistically in the attributable risk of both smoking and poor oral hygiene, it is conceivable to suggest a unifying pathogenetic mechanism of alcohol drinking behind these epidemiological findings. This could be the local production of carcinogenic acetaldehyde from ethanol by oral microbes.
There is increasing evidence for acetaldehyde to be the ultimate carcinogenic substance behind alcohol intake. Acetaldehyde has been shown to be highly toxic, mutagenic and carcinogenic in different cell cultures and animal models (7,2023). In experimental animals, histopathological changes after acetaldehyde treatment have been shown to mimic those known to occur after treatment with alcohol (24). Stronger evidence for acetaldehyde as the major factor behind ethanol-associated carcinogenesis is derived from studies linking the genotypes of ethanol-metabolizing enzymes with tumour risk. Rapid metabolizing ADHs (ADH3), leading to higher and quicker production of cellular acetaldehyde, and lack of the low Km aldehyde dehydrogenases (ALDHs) ALDH2, leading to a longer and delayed exposure to acetaldehyde, have recently been shown to be associated with increased cancer risk in the upper gastrointestinal tract (2529). In a very recent study among Orientals, a possible correlation between ALDH2 genotype mutation and cancer risk in alcoholics has been expanded to all possible alcohol-related cancers. In this study, the frequency of a mutant ALDH2-2 allele was significantly higher in alcoholics with oropharyngeal, laryngeal, oesophageal, stomach, colon and lung cancer, but not liver or other cancers (29). This is very interesting, as these organs are covered by microbes and microbial production of acetaldehyde from ethanol has been described (811,30). Thus, it is possible that the hampered detoxification of acetaldehyde from ethanol in ALDH2-deficient subjects might only become clinically relevant in cases of marked acetaldehyde production by microbes. Hence, there is conclusive experimental support for microbial acetaldehyde production from ethanol as a major factor in alcohol-associated carcinogenesis.
Recently, local acetaldehyde production in the saliva by microbes has been described (10). Microbial salivary acetaldehyde production shows high interindividual variation, but there exists a significant positive correlation between salivary ethanol and acetaldehyde levels. Moreover, in vivo salivary acetaldehyde levels correlate very significantly with the levels that are produced in vitro. This offers the opportunity to use the in vitro salivary test as a tool to investigate possible variables which might influence salivary acetaldehyde production. Salivary acetaldehyde levels after ethanol intake strikingly exceed those known to be derived from endogenous metabolism of ethanol (10). Salivary acetaldehyde may reach, via normal distribution and evaporation, all target tissues of the upper aerodigestive tract, such as the larynx, pharynx, oral cavity, oesophagus and even the stomach. Consequently, we suggest that the major part of the carcinogenic role of alcohol is caused by its first metabolite, acetaldehyde, which is microbially produced.
In the present study, we were able to demonstrate that smoking and heavy alcohol consumption significantly increase salivary acetaldehyde production. Smoking showed a positive linear correlation and it can be estimated that a smoker with a daily consumption of ~20 cigarettes has an increased salivary acetaldehyde production of ~5060%. This implies that smokers, even after moderate alcohol intake, produce much higher levels of carcinogenic acetaldehyde in the oral cavity than non-smokers. The evidence for increased microbial salivary acetaldehyde production in smokers, together with an epidemiological description of the multiplicative carcinogenic action of alcohol and smoking, suggests that salivary acetaldehyde production mediated by microbes could be a biologically plausible pathogenetic mechanism for these findings. Alcohol seems to interact and increase salivary acetaldehyde production only if consumption is heavy (>40 g/day); when an increase is observed it is dose dependent. Smoking and alcohol together increase salivary acetaldehyde production by ~100% as compared with non-smokers and moderate alcohol consumers.
It has been demonstrated that smoking and alcohol intake affect and slightly decrease saliva flow (31). It is well known that in the presence of a low saliva flow bacterial concentrations increase, and this could be the explanation for the observed higher total counts in these subjects. However, there are obviously also qualitative changes in the microbial flora in high acetaldehyde producers, as has already been described for smokers (3240). For instance, an increase in yeast infections, e.g. Candida albicans, has been demonstrated for smokers (3537). This is in accordance with our finding of an increased incidence and also higher loads of yeasts among high acetaldehyde producers. On the other hand, Neisseriae spp. have been reported to occur less frequently in the oral cavity of smokers and this species is not associated with higher acetaldehyde production (38,39). In general, a microbial `switch' with a significant increase in the proportion of Gram-positive versus Gram-negative bacteria has been described in smokers (34,3840). This is in line with our observation that almost all aerobic Gram-positive bacteria were significantly increased in `high' acetaldehyde producers (with the facultative commensals Staphylococcus sp. and S.mutans as the only exceptions), whereas the Gram-negative aerobic bacteria Haemophilus sp. and the already mentioned Neisseria sp. were not associated with higher acetaldehyde production. Thus, there is in general a good link between our microbial observations that some species are associated with higher acetaldehyde production and the well-known effect of smoking on the oral microflora.
Microbial changes in the oral microflora of alcoholics have been less intensively described (17,41). Epidemiological studies have shown that heavy drinking is associated with poor oral hygiene. It has been suggested that this may lead to bacterial overgrowth, but so far no study has convincingly proved this hypothesis and no bacterial species have been associated with high alcohol consumption (17). As high salivary acetaldehyde production was observed only among heavy drinkers, enzyme induction might be an explanation for this finding. Bacteria are known to be easily able to induce the corresponding metabolizing enzymes, however, it remains speculative whether this effect accounts for our observations.
Poor oral health status is a weak risk factor for oral cavity cancer (1416). Bacterial overgrowth and, in the case of concomitant alcohol intake, high bacterial acetaldehyde production have been suggested as pathogenetic mechanisms. Our study could not confirm these suggestions and the only indirect parameter of oral health which was weakly associated with higher acetaldehyde production was the self-reported frequency of dry mouth, which again could lead to higher bacterial loads per millilitre due to decreased flow. However, it can be questioned how reliably a self-reported questionnaire takes into account the factors representing actual oral status. The question whether salivary microbial acetaldehyde production might be the mechanism behind the higher cancer risk in alcoholics with poor oral hygiene remains unanswered and further studies that include actual dental status and a control for confounding factors are needed.
In the microbial fermentation of glucose to alcohol the conversion from acetaldehyde to ethanol is catalysed by ADHs and the reaction is reversible. Thus, in the case of substrate (ethanol) excess and in the presence of oxygen, the reaction runs in the opposite direction, with acetaldehyde as the end product. In this study, the total anaerobic counts included both facultative and microaerophilic bacteria that can grow under anaerobic conditions, but otherwise no anaerobic species were associated with higher acetaldehyde production. On the other hand, our microbial analyses revealed some Gram-positive aerobic bacterial strains and yeasts which where found at higher loads among high acetaldehyde producers. Moreover, yeasts (and possibly Corynebacterium sp.) were also found more frequently in this group. Recent experience with intestinal bacteria indicate that there are some strains which have a much higher acetaldehyde production capacity and ADH activity than others (30). Thus, our findings can be regarded as a first hint of high acetaldehyde-producing microbes. Moreover, our findings support the hypothesis that bacteria and yeasts are involved in ethanol-associated carcinogenesis and may represent the main metabolic source for the production of highly carcinogenic acetaldehyde from ethanol. This may open a new microbiological approach to the pathogenesis of the oral cavity and upper gastrointestinal tract cancer.
In contrast to our previous findings, we could not demonstrate a higher acetaldehyde production in patients with a malignancy of the oral cavity (11). First, saliva, and not mouthwash, was used as the probe in this study and we were able to demonstrate recently that in vitro salivary acetaldehyde is the method of choice to reliably reflect the acetaldehyde levels in vivo. Secondly, in this study a multivariate analysis was performed among 326 volunteers, whereas the number of patients in our previous study was limited (n = 53) and only univariate statistical analysis could be performed. Thirdly, controls and cancer patients were well balanced in our study with respect to alcohol consumption and smoking, whereas these risk factors were twice as high in the cancer cohort of our previous study (11). Thus, it is possible that our previous findings of higher acetaldehyde production among cancer patients might just have reflected the higher intake of tobacco products and alcohol in the cancer cohort rather than increased acetaldehyde production by the tumour or its influence on the microflora itself.
In conclusion, we have demonstrated increased microbial salivary acetaldehyde production in smokers and heavy drinkers. Numerous studies support the hypothesis that acetaldehyde is the substance behind the tumour-promoting effect of alcohol on the mucosa of the oral cavity. Salivary microbial production is supposed to be one of the major sources of acetaldehyde from ethanol. Thus, our finding could be a biologically plausible mechanism to explain the synergistic and multiplicative manner by which the attributable cancer risks of alcohol and smoking act. Several bacteria and yeasts have been found in significantly higher numbers in the saliva among high acetaldehyde producers, which offers a new microbiological approach to the pathogenesis of alcohol-associated carcinogenesis.
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Acknowledgments |
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Notes |
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References |
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