Interaction of alcohol and smoking in the pathogenesis of upper digestive tract cancers - possible chemoprevention with cysteine

Background: Alcohol consumption and smoking are the main causes of upper digestive tract cancers. These risk factors account for over 75% of all cases in developed countries. Epidemiological studies have shown that alcohol and tobacco interact in a multiplicative way to the cancer risk, but the pathogenetic mechanism behind this is poorly understood. Strong experimental and human genetic linkage data suggest that acetaldehyde is one of the major factors behind the carcinogenic effect. In the digestive tract, acetaldehyde is mainly formed by microbial metabolism of ethanol. Acetaldehyde is also a major constituent of tobacco smoke. Thus, acetaldehyde from both of these sources may have an interacting carcinogenic effect in the human upper digestive tract. Aims: The first aim of this thesis was to investigate acetaldehyde production and exposure in the human mouth resulting from alcohol ingestion and tobacco smoking in vivo. Secondly, specific L-cysteine products were prepared to examine their efficacy in the binding of salivary acetaldehyde in order to reduce the exposure of the upper digestive tract to acetaldehyde. Methods: Acetaldehyde levels in saliva were measured from human volunteers during alcohol metabolism, during tobacco smoking and during the combined use of alcohol and tobacco. The ability of L-cysteine to eliminate acetaldehyde during alcohol metabolism and tobacco smoking was also investigated with specifically developed tablets. Also the acetaldehyde production of Escherichia coli - an important member of the human microbiota - was measured in different conditions prevailing in the digestive tract. Results and conclusions: These studies established that smokers have significantly increased acetaldehyde exposure during ethanol consumption even when not actively smoking. Acetaldehyde exposure was dramatically further increased during active tobacco smoking. Thus, the elevated aerodigestive tract cancer risk observed in smokers and drinkers may be the result of the increased acetaldehyde exposure. Acetaldehyde produced in the oral cavity during ethanol challenge was significantly decreased by a buccal L-cysteine -releasing tablet. Also smoking-derived acetaldehyde could be totally removed by using a tablet containing L-cysteine. In conclusion, this thesis confirms the essential role of acetaldehyde in the pathogenesis of alcohol- and smoking-induced cancers. This thesis presents a novel experimental approach to decrease the local acetaldehyde exposure of the upper digestive tract with L-cysteine, with the eventual goal of reducting the prevalence of upper digestive tract cancers.

Probiotics in the prevention of clinical manifestations of common infectious diseases in children and in the elderly

Infectious diseases put an enormous burden on both children and the elderly in the forms of respiratory, gastrointestinal and oral infections. There is evidence suggesting that specific probiotics may be antagonistic to pathogens and may enhance the immune system, but the clinical evidence is still too sparce to make general conclusions on the disease-preventive effects of probiotics. This thesis, consisting of four independent, double-blind, placebo-controlled clinical trials, investigated whether Lactobacillus GG (LGG) or a specific probiotic combination containing LGG would reduce the risk of common infections or the prevalence of pathogens in healthy and infection-prone children and in independent and institutionalised elderly people. In healthy day-care children, the 7-month consumption of probiotic milk containing Lactobacillus GG appeared to postpone the first acute respiratory infection (ARI) by one week (p=0.03, adjusted p=0.16), and to reduce complicated infections (39% vs. 47%, p<0.05, adjusted p=0.13), as well as the need for antibiotic treatment (44% vs. 54%, p=0.03, adjusted p=0.08) and day-care absences (4.9 vs. 5.8 days, p=0.03, adjusted p=0.09) compared to the placebo milk. In infection-prone children, the 6-month consumption of a combination of four probiotic bacteria (LGG, L. rhamnosus LC705, Propionibacterium freudenreichii JS, Bifidobacterium breve 99) taken in capsules appeared to reduce recurrent ARIs (72% vs. 82%, p<0.05; adjusted p=0.06), and the effect was particularly noticeable in a subgroup of children with allergic diseases (12% vs. 33%, p=0.03), although no effect on the presence of nasopharyngeal rhinovirus or enterovirus was seen. The 5-month consumption of the same probiotic combination did not show any beneficial effects on the respiratory infections in frail, institutionalised elderly subjects. In healthy children receiving Lactobacillus GG, the reduction in complications resulted in a marginal reduction in the occurrence of acute otitis media (AOM) (31% vs. 39%, p=0.08; adjusted p=0.19), and the postponement of the first AOM episode by 12 days (p=0.04; adjusted p=0.09). However, in otitis-prone children, a probiotic combination did not reduce the occurrence of AOM or the total prevalence of common AOM pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis), except in the case of children with allergic diseases, in whom probiotics reduced recurrent AOM episodes (0% vs. 14%, p=0.03). In addition, interaction between probiotics and bacterial carriage was seen: probiot-ics reduced AOM in children who did not carry any bacterial pathogens (63% vs. 83%), but the effect was the reverse in children carrying bacteria in the nasopharynx (74% vs 62%) (p<0.05). Long-term probiotic treatment, either LGG given in milk to healthy children for 7 months or a combination of probiotics given in capsules to institutionalised elderly subjects for 5 months, did not reduce the occurrence of acute diarrhoea. However, when the probiotic combination (LGG, L. rhamnosus LC705, Propionibacterium JS) was given in cheese to independent elderly subjects for 4 months, the oral carriage of high Candida counts was reduced in the probiotic group vs. the placebo group (21% vs. 34%, p=0.01, adjusted p=0.004). The risk of hyposalivation was also reduced in the probiotic group (p=0.05). In conclusion, probiotics appear to slightly alleviate the severity of infections by postponing their appearance, by reducing complications and the need for antimicrobial treatments. In addition, they appear to prevent recurrent infections in certain subgroups of children, such as in infection-prone children with allergic diseases. Alleviating ARI by probiotics may lead to a marginal reduction in the occurrence of AOM in healthy children but not in infection-prone children with disturbed nasopharyngeal microbiota. On the basis of these results it could be supposed that Lactobacillus GG or a specific combination containing LGG are effective against viral but not against bacterial otitis, and the mechanism is probably mediated through the stimulation of the immune system. A specific probiotic combination does not reduce respiratory infections in frail elderly subjects. Acute diarrhoea, either in children or in the elderly, is not prevented by the continuous, long-term consumption of probiotics, but the consumption of a specific probiotic combination in a food matrix is beneficial to the oral health of the elderly, through the reduction of the carriage of Candida.

Oral health, smoking and adolescence

The present cross-sectional study examined the effect of smoking on oral health in a birth cohort of 15 to 16-year-old Finnish adolescents. The hypothesis was that oral health parameters were poorer among smoking than non-smoking subjects and that a tobacco intervention program could be effective among the adolescents. The study was conducted in the Kotka Health Center, Kotka, Finland. Altogether 501 out of 545 subjects (15- to 16-year-old boys [n = 258] and girls [n = 243]) were clinically examined in 2004 and 2005. The sample frame was a birth cohort of all subjects in 1989 and 1990, living in Kotka. A structured questionnaire was also filled in by the participants to record their general health and health habits, such as smoking, tooth brushing, and medication used. The participants were classified into nonsmokers, current smokers, and former smokers. Subgingival pooled plaque samples were taken and stimulated salivary samples were also collected. The subjects were asked from which of seven professional groups (doctors, school nurses, dental nurses, general nurses, dentists, teachers and media professionals) they would prefer to receive information about tobacco. The two most popular groups they picked up were dentists and school nurses. Current smokers (n=127) were then randomly assigned into three groups: the dentist group (n =44), the school-nurse group (n =42), and the control group (n =39). The intervention was based on a national recommendation of evidence based guidelines by The Finnish Medical Society Duodecim ( 5A counseling system). Two months after the intervention, a second questionnaire was sent to the smokers in the intervention groups. Smoking cessation, smoking quantity per week, and self-rated addiction for smoking (SRA) were recorded. The results were analyzed using the R-statistical program. The results showed that 15% of the subjects had periodontitis. Smokers (25%) had more periodontitis than non-smokers (66%) (p < 0.001). Smoking boys (24%) also had more caries lesions than non-smokers (69%) (p < 0.001), and they brushed their teeth less frequently than non-smokers. Smoking significantly impaired periodontal health of the subjects, even when the confounding effects of plaque and tooth brushing were adjusted. Smoking pack-years, intensified the effects of smoking. Periodontal bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia and Treponema denticola were more frequently detected among the smokers than non-smokers, especially among smoking girls. Smoking significantly decreased the values of both the salivary periodontal biomarkers MMP-8 (p=0.04) and PMN elastase (p=0.02) in boys. The effect was strengthened by pack years of smoking (MMP-8 p=0.04; elastase p0.01). Of those who participated in the intervention, 19 % quit smoking. The key factors associated with smoking cessation were best friend`s influence, nicotine dependence and diurnal type. When the best friend was not a smoker, the risk ratio (RR) of quit smoking after the intervention was 7.0 (Cl 95% 4.6 10.7). Of the diurnal types, the morning people seemed to be more likely to quit (RR 2.2 [Cl 95% 1.4 3.6]). Nicotine dependence also elicited an opposite effect: those who scored between 3 and 5 dependence scores were less likely to quit. In conclusion, smoking appears to be a major etiological risk factor for oral health. However, the early signs of periodontal disease were mild in the subjects studied. Based on the opinions of the adolescent s, dental professionals may have a key position in their smoking cessation. The harmful effects of smoking on oral health could be used in counselling. Best friend`s influence, nicotine dependence and diurnal type, all factors associated with smoking cessation, should be taken more carefully into account in the prevention programs for adolescents.

Molekyylibiologisten menetelmien kehittäminen ihmisen suoliston sulfaattia pelkistävien bakteerien määrittämiseksi

Ihmisen ruuansulatuskanavan bakteeriston kehitys alkaa syntymästä, jolloin ensimmäiset bakteerit kansoittavat steriilin ruuansulatuskanavan. Bakteeristo kehittyy perimän, ympäristön ja varhaisen ruokavalion vaikutuksesta kohti monimuotoisempaa bakteeripopulaatiota. Aikuisen ruuansulatuskanavan normaalibakteeristo on varsin muuttumaton, mutta siihen vaikuttavat monet tekijät, kuten ikä, terveydentila, ruokavalio ja antibioottien käyttö. Bakteeriston koostumus vaihtelee ruuansulatuskanavan eri osissa ja bakteerimäärä kasvaa kohti paksusuolta, ollen paksusuolessa ja ulosteessa peräti 1010-1012 pmy/ml. Suurin osa ruuansulatuskanavan bakteereista on anaerobeja. Ruuansulatuskanavan bakteeristo vaikuttaa muun muassa suoliston kehittymiseen ja hiilihydraattien ja proteiinien hajotukseen sekä toimii osana immuunipuolustusta. Sulfaattia pelkistävät bakteerit (SRB) ovat monimuotoinen ryhmä pääosin anaerobisia bakteereita, jotka käyttävät aineenvaihdunnassaan elektronin vastaanottajana sulfaattia muuttaen sen lopulta sulfidiksi. SRB:t ovat sopeutuneet useisiin erilaisiin ympäristöihin. Niitä tavataan mm. vesistöjen sedimenteissä sekä ihmisen ruuansulatuskanavassa. Ihmisen ruuansulatuskanavassa on SRB:ta n. 105-108 pmy/g, ja niitä on löydetty erityisesti anaerobisista osista kuten suun ientaskuista ja paksusuolesta. SRB:t voivat olla haitaksi ruuansulatuskanavalle tuottamansa sulfidin vuoksi, joka esiintyy vesiliuoksessa vetysulfidina. Tämän on havaittu olevan toksista suoliston epiteelisoluille. Viimeaikoina on kiinnostuttu sulfaatinpelkistäjien yhteydestä suoliston sairaustiloihin, kuten tulehduksellisiin suolistosairauksiin (IBD). Pro gradu -tutkimukseni tavoitteena oli kehittää PCR-DGGE- ja qPCR-menetelmät ulosteen sulfaattia pelkistävien bakteerien määritykseen. Kohdegeeninä menetelmänkehityksessä käytettiin dsrAB-geeniä, joka koodaa dissimilatorista sulfiitinpelkistysentsyymiä. dsrAB-geeni on sulfaatinpelkistäjille ominainen konservoitunut geenialue, johon perustuvia tutkimuksia ei vielä ole paljon ihmispuolelta. qPCR-menetelmä saatiin optimoitua herkäksi ja spesifiseksi käyttäen dsrA-geenispesifisiä alukkeita, mutta PCR-DGGE-menetelmää ei saatu optimoitua käytössä olleilla alukkeilla, jotka monistivat PCR-DGGE:ssa myös negatiivikontrollikantoja. Tutkittaessa qPCR:lla IBD:tä (Crohn ja ulseratiivinen koliitti) sairastavien lasten ja terveiden kontrollihenkilöiden ulostenäytteistä eristettyä DNA:ta, merkittävää eroa SRB-määrissä ei havaittu eri ryhmien välillä. Crohnin tautia sairastavien aktiivisen vaiheen ja oireettoman vaiheen näytteiden välillä oli kuitenkin tilastollisesti merkitsevä ero (SRB-määrät; oireeton vaihe>oireellinen vaihe) (P <0,05).

Microbiological characterisation of soils : Evaluation of some critical steps in data collection and experimental design

The study of soil microbiota and their activities is central to the understanding of many ecosystem processes such as decomposition and nutrient cycling. The collection of microbiological data from soils generally involves several sequential steps of sampling, pretreatment and laboratory measurements. The reliability of results is dependent on reliable methods in every step. The aim of this thesis was to critically evaluate some central methods and procedures used in soil microbiological studies in order to increase our understanding of the factors that affect the measurement results and to provide guidance and new approaches for the design of experiments. The thesis focuses on four major themes: 1) soil microbiological heterogeneity and sampling, 2) storage of soil samples, 3) DNA extraction from soil, and 4) quantification of specific microbial groups by the most-probable-number (MPN) procedure. Soil heterogeneity and sampling are discussed as a single theme because knowledge on spatial (horizontal and vertical) and temporal variation is crucial when designing sampling procedures. Comparison of adjacent forest, meadow and cropped field plots showed that land use has a strong impact on the degree of horizontal variation of soil enzyme activities and bacterial community structure. However, regardless of the land use, the variation of microbiological characteristics appeared not to have predictable spatial structure at 0.5-10 m. Temporal and soil depth-related patterns were studied in relation to plant growth in cropped soil. The results showed that most enzyme activities and microbial biomass have a clear decreasing trend in the top 40 cm soil profile and a temporal pattern during the growing season. A new procedure for sampling of soil microbiological characteristics based on stratified sampling and pre-characterisation of samples was developed. A practical example demonstrated the potential of the new procedure to reduce the analysis efforts involved in laborious microbiological measurements without loss of precision. The investigation of storage of soil samples revealed that freezing (-20 °C) of small sample aliquots retains the activity of hydrolytic enzymes and the structure of the bacterial community in different soil matrices relatively well whereas air-drying cannot be recommended as a storage method for soil microbiological properties due to large reductions in activity. Freezing below -70 °C was the preferred method of storage for samples with high organic matter content. Comparison of different direct DNA extraction methods showed that the cell lysis treatment has a strong impact on the molecular size of DNA obtained and on the bacterial community structure detected. An improved MPN method for the enumeration of soil naphthalene degraders was introduced as an alternative to more complex MPN protocols or the DNA-based quantification approach. The main advantage of the new method is the simple protocol and the possibility to analyse a large number of samples and replicates simultaneously.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.

Production of Carcinogenic Acetaldehyde by Oral Microbiome

Oral cancer is the seventh most common cancer worldwide and its incidence is increasing. The most important risk factors for oral cancer are chronic alcohol consumption and tobacco smoking, up to 80 % of oral carcinomas are estimated to be caused by alcohol and tobacco. They both trigger an increased level of salivary acetaldehyde, during and after consumption, which is believed to lead to carcinogenesis. Acetaldehyde has multiple mutagenic features and it has recently been classified as a Group 1 carcinogen for humans by the International Agency for Research on Cancer. Acetaldehyde is metabolized from ethanol by microbes of oral microbiota. Some oral microbes possess alcohol dehydrogenase enzyme (ADH) activity, which is the main enzyme in acetaldehyde production. Many microbes are also capable of acetaldehyde production via alcohol fermentation from glucose. However, metabolism of ethanol into acetaldehyde leads to production of high levels of this carcinogen. Acetaldehyde is found in saliva during and after alcohol consumption. In fact, rather low ethanol concentrations (2-20mM) derived from blood to saliva are enough for microbial acetaldehyde production. The high acetaldehyde levels in saliva after alcohol challenge are explained by the lack of oral microbiota and mucosa to detoxify acetaldehyde by metabolizing it into acetate and acetyl coenzymeA. The aim of this thesis project was to specify the role of oral microbes in the in vitro production of acetaldehyde in the presence of ethanol. In addition, it was sought to establish whether microbial metabolism could also produce acetaldehyde from glucose. Furthermore, the potential of xylitol to inhibit ethanol metabolism and acetaldehyde production was explored. Isolates of oral microbes were used in the first three studies. Acetaldehyde production was analyzed after ethanol, glucose and fructose incubation with gas chromatography measurement. In studies I and III, the ADH enzyme activity of some microbes was measured by fluorescence. The effect of xylitol was analyzed by incubating microbes with ethanol and xylitol. The fourth study was made ex vivo and microbial samples obtained from different patient groups were analyzed. This work has demonstrated that isolates of oral microbiota are able to produce acetaldehyde in the presence of clinically relevant ethanol and glucose concentrations. Significant differences were found between microbial species and isolates from different patient groups. In particular, the ability of candidal isolates from APECED patients to produce significantly more acetaldehyde in glucose incubation compared to healthy and cancer patient isolates is an interesting observation. Moreover, xylitol was found to reduce their acetaldehyde production significantly. Significant ADH enzyme activity was found in the analyzed high acetaldehyde producing streptococci and candida isolates. In addition, xylitol was found to reduce the ADH enzyme activity of C. albicans. Some results from the ex vivo study were controversial, since acetaldehyde production did not correlate as expected with the amount of microbes in the samples. Nevertheless, the samples isolated from patients did produce significant amounts of acetaldehyde with a clinically relevant ethanol concentration.