The Importance of Nitrate Reduction for Oral Health.

Salivary glands concentrate plasma nitrate into saliva, leading to high nitrate concentrations that can reach the millimolar range after a nitrate-rich vegetable meal. Whereas human cells cannot reduce nitrate to nitrite effectively, certain oral bacteria can. This leads to an increase in systemic nitrite that can improve conditions such as hypertension and diabetes through nitric oxide availability. Apart from systemic benefits, it has been proposed that microbial nitrate reduction can also promote oral health. In this review, we discuss evidence associating dietary nitrate with oral health. Oral bacteria can reduce nitrite to nitric oxide, a free radical with antimicrobial properties capable of inhibiting sensitive species such as anaerobes involved in periodontal diseases. Nitrate has also been shown to increase resilience against salivary acidification in vivo and in vitro, thus preventing caries development. One potential mechanism is proton consumption during denitrification and/or bacterial reduction of nitrite to ammonium. Additionally, lactic acid (organic acid involved in oral acidification) and hydrogen sulfide (volatile compound involved in halitosis) can act as electron donors for these processes. The nitrate-reducing bacteria Rothia and Neisseria are consistently found at higher levels in individuals free of oral disease (vs. individuals with caries, periodontitis, and/or halitosis) and increase when nitrate is consumed in clinical studies. Preliminary in vitro and clinical evidence show that bacteria normally associated with disease, such as Veillonella (caries) and Prevotella (periodontal diseases and halitosis), decrease in the presence of nitrate. We propose nitrate as an ecologic factor stimulating eubiosis (i.e., an increase in health-associated species and functions). Finally, we discuss the preventive and therapeutic potential, as well as safety issues, related to the use of nitrate. In vivo evidence is limited; therefore, robust clinical studies are required to confirm the potential benefits of nitrate reduction on oral health.

The benefits and risks of beetroot juice consumption: a systematic review.

Beetroot juice (BRJ) has become increasingly popular amongst athletes aiming to improve sport performances. BRJ contains high concentrations of nitrate, which can be converted into nitric oxide (NO) after consumption. NO has various functions in the human body, including a vasodilatory effect, which reduces blood pressure and increases oxygen- and nutrient delivery to various organs. These effects indicate that BRJ may have relevant applications in prevention and treatment of cardiovascular disease. Furthermore, the consumption of BRJ also has an impact on oxygen delivery to skeletal muscles, muscle efficiency, tolerance and endurance and may thus have a positive impact on sports performances. Aside from the beneficial aspects of BRJ consumption, there may also be potential health risks. Drinking BRJ may easily increase nitrate intake above the acceptable daily intake, which is known to stimulate the endogenous formation of N-nitroso compounds (NOC's), a class of compounds that is known to be carcinogenic and that may also induce several other adverse effects. Compared to studies on the beneficial effects, the amount of data and literature on the negative effects of BRJ is rather limited, and should be increased in order to perform a balanced risk assessment.

Dietary acrylamide intake and risk of breast cancer in the UK women's cohort.

BACKGROUND: No studies to date have demonstrated a clear association with breast cancer risk and dietary exposure to acrylamide. METHODS: A 217-item food frequency questionnaire was used to estimate dietary acrylamide intake in 33,731 women aged 35-69 years from the UK Women's Cohort Study followed up for a median of 11 years. RESULTS: In all, 1084 incident breast cancers occurred during follow-up. There was no evidence of an overall association between acrylamide intake and breast cancer (hazard ratio=1.08 per 10 µg day(-1), 95% CI: 0.98-1.18, P(trend)=0.1). There was a suggestion of a possible weak positive association between dietary acrylamide intake and premenopausal breast cancer after adjustment for potential confounders (hazard ratio=1.2, 95% CI: 1.0-1.3, P(trend)=0.008). There was no suggestion of any association for postmenopausal breast cancer (hazard ratio=1.0, 95% CI: 0.9-1.1, P(trend)=0.99). CONCLUSIONS: There is no evidence of an association between dietary acrylamide intake and breast cancer. A weak association may exist with premenopausal breast cancer, but requires further investigation.

Proton pump inhibitor therapy in gastro-oesophageal reflux disease decreases the oesophageal immune response but does not reduce the formation of DNA adducts.

BACKGROUND: Chronic oesophageal inflammation and related oxidative stress are important in the pathogenesis of erosive oesophagitis (EO) and its malignant progression. AIM: To study the effect of proton pump inhibitors (PPIs) on oesophageal cellular immune response and oxidative damage in EO patients. METHODS: Forty gastro-oesophageal reflux disease (GERD) patients [non-erosive reflux disease (NERD): 15, EO: 25] were included, after 7 days off antisuppressive drugs. EO patients were randomized to 20-mg rabeprazole once daily for either 4 or 8 weeks with baseline and follow-up endoscopy with distal oesophageal biopsies. T lymphocytes, macrophages and mast cells were quantified by immunohistochemistry. DNA adducts were measured by analysis of 8-oxo-deoxyguanosine levels. RESULTS: Erosive oesophagitis patients had more T lymphocytes and CD8(+) T lymphocytes in squamous epithelium than NERD patients (P = 0.001, P = 0.002, respectively). Levels of DNA adducts between both groups were, however, not different (P = 0.99). Four- and eight-week rabeprazole treatment in EO patients resulted in a significant decrease in number of T lymphocytes and CD8(+) T lymphocytes (all P < 0.05). PPIs did not, however, affect levels of DNA adducts. CONCLUSIONS: Short-term PPI therapy in EO patients reduces the oesophageal cellular immune response, but does not change oxidative damage. PPI therapy may therefore not be effective in reducing the risk of oesophageal cancer in GERD patients.

Comparison of supervised clustering methods to discriminate genotoxic from non-genotoxic carcinogens by gene expression profiling.

Prediction of the toxic properties of chemicals based on modulation of gene expression profiles in exposed cells or animals is one of the major applications of toxicogenomics. Previously, we demonstrated that by Pearson correlation analysis of gene expression profiles from treated HepG2 cells it is possible to correctly discriminate and predict genotoxic from non-genotoxic carcinogens. Since to date many different supervised clustering methods for discrimination and prediction tests are available, we investigated whether application of the methods provided by the Whitehead Institute and Stanford University improved our initial prediction. Four different supervised clustering methods were applied for this comparison, namely Pearson correlation analysis (Pearson), nearest shrunken centroids analysis (NSC), K-nearest neighbour analysis (KNN) and Weighted voting (WV). For each supervised clustering method, three different approaches were followed: (1) using all the data points for all treatments, (2) exclusion of the samples with marginally affected gene expression profiles and (3) filtering out the gene expression signals that were hardly altered. On the complete data set, NSC, KNN and WV outperformed the Pearson test, but on the reduced data sets no clear difference was observed. Exclusion of samples with marginally affected profiles improved the prediction by all methods. For the various prediction models, gene sets of different compositions were selected; in these 27 genes appeared three times or more. These 27 genes are involved in many different biological processes and molecular functions, such as apoptosis, cell cycle control, regulation of transcription, and transporter activity, many of them related to the carcinogenic process. One gene, BAX, was selected in all 10 models, while ZFP36 was selected in 9, and AHR, MT1E and TTR in 8. Summarising, this study demonstrates that several supervised clustering methods can be used to discriminate certain genotoxic from non-genotoxic carcinogens by gene expression profiling in vitro in HepG2 cells. None of the methods clearly outperforms the others.

Discrimination of genotoxic from non-genotoxic carcinogens by gene expression profiling.

Two general mechanisms are implicated in chemical carcinogenesis. The first involves direct damage to DNA, referred to as genotoxic (GTX), to which the cell responds by repair of the damages, arrest of the cell cycle or induction of apoptosis. The second is non-DNA damaging, non-genotoxic (NGTX), in which a wide variety of cellular processes may be involved. Therefore, it can be hypothesized that modulation of the underlying gene expression patterns is profoundly distinct between GTX and NGTX carcinogens, and thus that expression profiling is applicable for classification of chemical carcinogens as GTX or NGTX. We investigated this hypothesis by analysing modulation of gene expression profiles induced by 20 chemical carcinogens in HepG2 cells with application of cDNA microarrays that contain 597 toxicologically relevant genes. In total, 22 treatments were included, divided in two sets. The training set consisted of 16 treatments (nine genotoxins and seven non-genotoxins) and the validation set of six treatments (three and three). Class discrimination models based on Pearson correlation analyses for the 20 most discriminating genes were developed with data from the training set, where after the models were tested with all data. Using all data, the correctness for classification of the carcinogens from the training set was clearly better than that for the validation set, namely 81 and 33%, respectively. Exclusion of the treatments that had only marginal effects on the expression profiles, improved the discrimination for the training and validation sets to 92 and 100% correctness, respectively. Exclusion of the gene expression signals that were hardly altered also improved classification, namely to 94 and 80%. Therefore, our study proves the principle that gene expression profiling can discriminate carcinogens with major differences in their mode of actions, namely genotoxins versus non-genotoxins.

Applicability of induced sputum for molecular dosimetry of exposure to inhalatory carcinogens: 32P-postlabeling of lipophilic DNA adducts in smokers and nonsmokers.

The lung is a major target organ for smoking-associated cancer. We examined the applicability of induced sputum for molecular dosimetry of exposure to tobacco smoke-related carcinogens. Sputum induction was performed by inhalation of 4.5% saline delivered from an ultrasonic nebulizer for a period of up to 21 min in a group of smoking (n = 20) and nonsmoking (n = 24) healthy individuals. Samples were analyzed for total and differential cell counts and cell viability. Subsequently, DNA contents of the samples were isolated, and measurement of lipophilic DNA adducts was done by the 32P-postlabeling assay using nuclease P1 (NP1) and butanol enrichment methods. All subjects tolerated the induction procedure without experiencing any troublesome symptoms, and 90% of smokers (18 of 20) and 88% of nonsmokers (21 of 24) succeeded in producing sufficient amounts of sputum. Total cell counts and percentages of viable cells in smokers were higher than those in nonsmokers (6.7+/-6.0 versus 4.7+/-6.0 x 10(6), P = 0.40 and 80+/-15 versus 63+/-17, P = 0.01, respectively). In cell differentials, smokers had lower percentages of bronchoalveolar macrophages and higher percentages of neutrophils (69+/-24 versus 92+/-5, P = 0.002 and 26+/-26 versus 4+/-4, P = 0.008, respectively). Using the NP1 digestion method, all smokers and only one nonsmoker showed a diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (3.1+/-1.4 versus 0.6+/-0.8/10(8) nucleotides; P = 0.0007), and also, adduct levels were significantly related to smoking indices. Applying the butanol extraction method, however, only half of the smokers and three nonsmokers showed the diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (4.6+/-3.7 versus 1.0+/-1.9/10(8) nucleotides; P = 0.02), and the levels of adducts were significantly related to the smoking indices. There was a correlation between the levels of adducts determined by the two enrichment methods (r = 0.7; P = 0.02). Paired comparison showed no differences between the levels of adducts measured by the two methods (P = 0.55). We conclude that induced sputum can serve for molecular dosimetry of inhalatory exposure to carcinogens and that the NP1 version of the 32P-postlabeling assay is a choice of preference for studying smoking-induced DNA adducts in the lower respiratory tract.

Does the risk of childhood diabetes mellitus require revision of the guideline values for nitrate in drinking water?

In recent years, several studies have addressed a possible relationship between nitrate exposure and childhood type 1 insulin-dependent diabetes mellitus. The present ecologic study describes a possible relation between the incidence of type 1 diabetes and nitrate levels in drinking water in The Netherlands, and evaluates whether the World Health Organization and the European Commission standard for nitrate in drinking water (50 mg/L) is adequate to prevent risk of this disease. During 1993-1995 in The Netherlands, 1,104 cases of type 1 diabetes were diagnosed in children 0-14 years of age. We were able to use 1,064 of these cases in a total of 2,829,020 children in this analysis. We classified mean nitrate levels in drinking water in 3,932 postal code areas in The Netherlands in 1991-1995 into two exposure categories. One category was based on equal numbers of children exposed to different nitrate levels (0.25-2.08, 2.10-6.42, and 6.44-41.19 mg/L nitrate); the other was based on cut-off values of 10 and 25 mg/L nitrate. We determined standardized incidence ratios (SIRs) for type 1 diabetes in subgroups of the 2,829,020 children with respect to both nitrate exposure categories, sex, and age and as compared in univariate analysis using the chi-square test for trend. We compared the incidence rate ratios (IRRs) by multivariate analysis in a Poisson regression model. We found an effect of increasing age of the children on incidence of type 1 diabetes, but we did not find an effect of sex or of nitrate concentration in drinking water using the two exposure categories. For nitrate levels > 25 mg/L, an increased SIR and an increased IRR of 1.46 were observed; however, this increase was not statistically significant, probably because of the small number of cases (15 of 1,064). We concluded that there is no convincing evidence that nitrate in drinking water at current exposure levels is a risk factor for childhood type 1 diabetes mellitus in The Netherlands, although a threshold value > 25 mg/L for the occurrence of this disease can not be excluded.