Nitric oxide (NO) has led a revolution in physiology and pharmacology research during the last two decades. This labile molecule plays an important role in many functions in the body regulating vasodilatation, blood flow, mitochondrial respiration and platelet function. Currently, it is known that NO synthesis occurs via at least two physiological pathways: NO synthase (NOS) dependent and NOS independent. In the former, L-arginine is the main precursor. It is widely recognized that this amino acid is oxidized to NO by the action of the NOS enzymes. Additionally, L-citrulline has been indicated to be a secondary NO donor in the NOS-dependent pathway, since it can be converted to L-arginine. Nitrate and nitrite are the main substrates to produce NO via the NOS-independent pathway. These anions can be reduced in vivo to NO and other bioactive nitrogen oxides. Other molecules, such as the dietary supplement glycine propionyl-L-carnitine (GPLC), have also been suggested to increase levels of NO, although the physiological mechanisms remain to be elucidated. The interest in all these molecules has increased in many fields of research. In relation with exercise physiology, it has been suggested that an increase in NO production may enhance oxygen and nutrient delivery to active muscles, thus improving tolerance to physical exercise and recovery mechanisms. Several studies using NO donors have assessed this hypothesis in a healthy, trained population. However, the conclusions from these studies showed several discrepancies. While some reported that dietary supplementation with NO donors induced benefits in exercise performance, others did not find any positive effect. In this regard, training status of the subjects seems to be an important factor linked to the ergogenic effect of NO supplementation. Studies involving untrained or moderately trained healthy subjects showed that NO donors could improve tolerance to aerobic and anaerobic exercise. However, when highly trained subjects were supplemented, no positive effect on performance was indicated. In addition, all this evidence is mainly based on a young male population. Further research in elderly and female subjects is needed to determine whether NO supplements can induce benefit in exercise capacity when the NO metabolism is impaired by age and/or estrogen status.

Following a single blind, cross-over and non-randomized design we investigated the effect of 7-day use of chlorhexidine (CHX) mouthwash on the salivary microbiome as well as several saliva and plasma biomarkers in 36 healthy individuals. They rinsed their mouth (for 1 min) twice a day for seven days with a placebo mouthwash and then repeated this protocol with CHX mouthwash for a further seven days. Saliva and blood samples were taken at the end of each treatment to analyse the abundance and diversity of oral bacteria, and pH, lactate, glucose, nitrate and nitrite concentrations. CHX significantly increased the abundance of Firmicutes and Proteobacteria, and reduced the content of Bacteroidetes, TM7, SR1 and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity, accompanied by an increase in saliva lactate and glucose levels. Lower saliva and plasma nitrite concentrations were found after using CHX, followed by a trend of increased systolic blood pressure. Overall, this study demonstrates that mouthwash containing CHX is associated with a major shift in the salivary microbiome, leading to more acidic conditions and lower nitrite availability in healthy individuals.Chlorhexidine (CHX) has been commonly used in dental practice as antiseptic agent since 1970, due to its long-lasting antibacterial activity with a broad-spectrum of action 1 . Since then, many clinical trials have shown effective results of CHX for the clinical management of dental plaque and gingival inflammation and bleeding 2-4 . This is supported by other studies using in vitro methods and reporting positive results of CHX in reducing the proliferation of bacterial species associated with periodontal disease, such as Enterobacteria, Porphyromonas gingivalis, Fusobacterium nucleatum, as well as different species of Actinomyces and Streptococcus, including Streptococcus mutans, which is considered the main etiological agent of dental caries 4,5 . Other studies have also reported that the use of CHX was effective in the treatment of halitosis, especially in reducing the levels of halitosis-related bacteria colonising the dorsal surface of the tongue 6 .The anti-microbial activity of CHX however, has been extensively studied using in vitro culture methods, which limit the identification and cultivation of all microorganisms in the environment 4 . To the best of our knowledge, only one recent study has investigated the effect of CHX mouthwash on mixed bacterial communities (microbiome) of the tongue using new genome sequencing techniques such as 16 S rRNA 7 . The study found differences in over 10 different species colonizing the tongue, and a lower microbial diversity after using CHX for a week, but did not analyse other parameters related to oral health such as pH, lactate production or buffering capacity 7 . Additionally, we and others have recently shown that the use of CHX in healthy subjects can attenuate the nitrate-reducing activity of oral bacteria by at least 80% 8-11 . This in turn leads to lo...

Objectives Chlorhexidine (CHX) is a commonly used antiseptic mouthwash, used by dental practitioners and the public, due to its antimicrobial effects. The aim of this article was to provide a narrative review of current antimicrobial uses of CHX relevant to dentistry in the context of oral diseases, highlighting need for further studies to support its safe and appropriate use. Study selection, data and sources Randomised controlled trials, systematic reviews and national (UK and US) guidelines were consulted where available, with search terms for each subject category entered into MEDLINE, PubMed, Google Scholar and the Cochrane database. Results Some evidence existed to support adjunctive short-term use of CHX to manage dental plaque, and reduce clinical symptoms of gingivitis, dry socket, as well as reduce aerosolisation of bacteria. However, use must be weighed alongside the less desirable effects of CHX, including extrinsic staining of teeth, antimicrobial resistance to antiseptic agents and the rare, but fatal, allergic reactions to CHX. Conversely, evidence for the effectiveness of chlorhexidine to manage or prevent periodontitis, dental caries, necrotising periodontal diseases, peri-implantitis, and infections associated with extraction and aerosolised viruses remains less certain. Conclusions The use of CHX in dentistry and oral healthcare continues to be widespread and thus it is important that dental practitioners understand that, based on its differential mechanisms of action on different microbes, appropriate clinical and dental use of CHX should be oral disease specific. However, further scientific and clinical research is required before full recommendations can be made.