# Salivary Extracellular DNA and DNase Activity in Periodontitis
## 1 Introduction  
Extracellular DNA (ecDNA), i.e., DNA outside of the cells, was first discovered in the plasma of pregnant women [1] and cancer patients [2]. EcDNA could trigger inflammation and worsen the state of patients during pregnancy complications or cancer. In plasma, ecDNA has been studied also asa potential biomarker of sepsis [3], trauma [4], diabetes mellitus or organ transplantation [5]. It has been hypothesized that pragmatic ecDNA could be used as a personalized cancer therapy [6]. Even though usually plasma serves as a source of ecDNA, despite the common difficulties, such as high fragmentation and high variability in the yield, saliva might be of special interest in oral diseases, since it reflects the local oral conditions. Also, ecDNA has been studied in saliva of head and neck cancer patients, and it has been shown that mitochondrial DNA concentration is increased in saliva in these patients when compared to control healthy group [7]. All of the above indicates that body fluids DNA or ecDNA are not a disease-specific marker, however, it points towards pathogeneses of diseases, which is not fully understood.  
Periodontitis is a chronic multifactorial inflammatory oral disease. It starts with bacterial plaque concentrating near the gingival part followed by gingivitis. The oral microbiome indeed plays a role in this initial stage [8]. Under normal circumstances, there is a fragile balance between microbes that act as the nonharmful commensals and the oral health status [9]. The dysbiosis consequence includes plaque build-up consisting of immunogenic substances in the subgingival crevice of teeth, which leads to the further activation of the immune system. The resulting inflammation and neutrophils activation, as a nonspecific reaction, damages the surrounding healthy tissue releasing several other immunogenic substances, triggering thus immune overreaction and circulus vitiosus. In susceptible individuals, the untreated inflammation can later outgrow into loss of connective tissue and decline in bone support (periodontitis) and eventually a tooth loss [10; 11] with permanent inflammation of surrounding tissue. Recently, neutrophil extracellular traps as welbike structures containing DNA were already described as potential immunogenic agents in periodontitis, and they were hypothesized to be involved in the pathogenesis of this disease [12; 13; 14]. Neutrophils are activated during inflammation, directed towards the location of inflammation, in our case the oral cavity, and the fight against bacteria forming the traps. Neutrophil extracellular traps consist of histone proteins and DNA, therefore they are one of the various sources of ecDNA among others such as apoptosis or necrosis [15]. Salivary ecDNA obtained by double-centrifugation, as well as DNA in whole and centrifuged saliva, as potential triggers of inflammation in periodontitis, are worth testing as possible indicators of the oral status. As in plasma, there are also nucleases in saliva, such as deoxyribonuclease (DNase) that is responsible for degradation of ecDNA [16; 17]. Therefore, we think that, together with DNA concentrations and origin analysis, it is also important to specify activity of DNase in saliva. We hypothesize that salivary DNA will be increased in patients with worsened oral health and DNase activity could be also altered. Therefore, in this study, we investigated whether DNA in whole saliva, in centrifuged and double-centrifuged samples differs between periodontitis and healthy controls and whether salivary DNase activity is affected.