Maintaining proper oral hygiene is crucial for maintenance of overall oral health [1]. The oral cavity is the first entry point to the body, so the oral microbiome is constantly being influenced by daily activities such as eating, brushing, friction, and habits like smoking or drinking alcohol, as well as, antiseptic mouthwash use [2]. Recent data suggest that the global mouthwash market is projected to experience steady growth, with an estimated compound annual growth rate (CAGR) of 4.6% between 2025 and 2030. This reflects the widespread use of mouthwash as part of daily oral care routines [3-6]. Several recent observational surveys from Saudi Arabia have reported mouthwash use among ranging from 30 to 80% of the population as part of common oral hygiene practices [7-9]. Healthy individuals often use mouthwash to freshen their breath or as part of their general health and wellness routine [10]. In addition, antiseptic mouthwash is widely used among hospitalized patients to support the maintenance, or improvement, of their oral health [11,12]. However, emerging evidence suggests that routine use of antiseptic mouthwash may have detrimental systemic effects by disrupting the oral microbiome responsible for nitrate (NO3) reduction and nitric oxide (NO) production [13,14].   NO is a signaling molecule that plays a vital role in numerous biological processes, such as regulating vascular tone, supporting neurotransmission, and maintaining muscle energy metabolism [15]. Sufficient NO production is crucial for the maintenance of a healthy vascular system [16]. NO can be generated endogenously through the classic NO synthase-dependent pathway, which synthesize NO from l-arginine and oxygen in presence several co-factors. In addition, NO can be generated through the alternative pathway by the enzyme-independent entero-salivary circulation [16]. This exogenous pathway is called nitrate-nitrite-NO (NO3⁻ – NO2⁻ – NO) pathway. Dietary NO₃⁻, which is abundant in beetroot and green leafy vegetables, serves as a significant source of NO through the non-enzymatic pathway, where oral bacteria first reduce NO₃⁻ to NO2 , which is then further converted to NO under acidic and low-oxygen conditions [17]. Subsequently, the remaining NO2- pass to the intestines and are systemically absorbed, where it can be further reduced to NO. Given that a substantial proportion of circulating NO₂⁻ originates from dietary intake, the combination of dietary NO₃⁻ and oral bacterial activity plays a critical role in maintaining NO homeostasis and may serve as a vital defense against conditions linked to NO deficiency [17].   Some publications discuss and support the use of antiseptic mouthwash [18] such as chlorhexidine, including studies from Saudi Arabia [19], to enhance oral hygiene. However, these papers often overlook potential systemic effects. The use of a 0.12% chlorhexidine antiseptic mouthwash has been shown to substantially impair dietary NO3⁻ metabolism [20]. Research indicates that such mouthwash can eliminate up to 94% of the oral bacteria involved in NO3⁻ reduction, leading to an approximate 85% reduction in the conversion of NO3⁻- to NO2⁻ [21]. This disruption significantly compromises NO production and limits the health benefits usually associated with dietary NO3⁻ intake such as lowering blood pressure [22]. Furthermore, recent findings also show that NO3⁻ intake can beneficially modulate the oral microbiome by enriching NO3⁻reducing bacteria, further supporting NO bioavailability and contributing to local anti-inflammatory effects in the oral cavity [23]. Therefore, the widespread promotion of mouthwash use without proper clarification of its possible systemic implications may unintentionally contribute to public health risks, particularly concerning vascular function and blood pressure regulation.   Given the essential role of oral bacteria in maintaining systemic NO levels and supporting cardiovascular health, awareness of these mechanisms among dental healthcare professionals is crucial. A recent cross-sectional study conducted among dental practitioners in the Qassim region, Saudi Arabia found that a large proportion of practitioners (46.7%) routinely recommended the use of mouthwash twice daily, with 71.7% advising its use after toothbrushing [24]. Therefore, the current study aimed to evaluate Saudi dental professionals’ and dental students’ knowledge about the importance of NO and its derivatives and the role of oral bacteria and the use of antiseptic mouthwash in NO production. Additionally, the study examined whether knowledge levels differed based on participants’ gender, age, years of experience, professional qualification, and region of practice within Saudi Arabia. Understanding these potential adverse effects is essential for dental professionals to make informed decisions when recommending mouthwash products to their patients.

Study Design and Participants A cross-sectional study was undertaken across various regions in Saudi Arabia to assess the knowledge of Saudi dental professionals and students regarding the significance of NO and its derivatives, as well as the role of oral bacteria and antiseptic mouthwash use in NO production. Participants were recruited using a convenience sampling method and included dental students and practitioners recruited from the public and private dental sectors, as well as dental professionals working in academic settings.   Questionnaire Development and Validation The questionnaire was initially drafted by the principal investigator and refined with input two subject-matter experts to ensure clarity and content validity. Face validation involved six experts in dentistry and nutrition, who assessed the questionnaire for clarity, logic, and relevance. A pilot study with 12 respondents was then conducted to evaluate the clarity and comprehension of the questions. Based on this preliminary testing, necessary revisions were made to improve the suitability and understandability of the survey instrument. A summary of these steps is provided in Figure1.   The questions focused on knowledge and understanding among dental professionals and dental students about the importance of NO in health, as well as the effects of oral bacteria and using antiseptic mouthwash on dietary NO3⁻ metabolism and NO pathway. The questionnaire consisted of three sections: (1) demographic information (gender, age, education level, years of experience, work setting, and region of practice); (2) general knowledge about NO, assessed by seven questions covering awareness, physiological roles, and dietary sources of NO; and (3) specific knowledge regarding the role of oral bacteria, the effects of antiseptic mouthwash, and the NO₃⁻ – NO₂⁻–NO pathway, assessed by four questions respectively.   The general knowledge section began with an introductory question regarding participants’ prior awareness of NO, whether participants had heard about NO. This was followed by 7 scored questions that assessed basic knowledge about NO including potential confusion with nitrous oxide, endogenous production of NO, its physiological roles in the human body, effects on cardiovascular health and immune function, association with periodontal disease, and the ability of the body to synthesize NO from dietary sources. An additional unscored question assessed familiarity with the NO3⁻ – NO2⁻ – NO pathway and its relevance to health.   The specific knowledge section explored participants' understanding of the role of oral bacteria in NO production, the impact of antibacterial mouthwash on NO levels. Additionally, two questions were included that focused on participants’ opinions regarding the link between antibacterial mouthwash and cardiovascular disease, and on whether antibacterial toothpaste has a different effect on NO production compared to mouthwash. Questions were multiple-choice or yes/no/I don’t know.   Each correct answer scored 1 point; incorrect or "I don’t know" responses scored 0. The final questionnaire was distributed online, in English, between January 1st and April 24th, 2025. Recruitment utilized professional association mailing lists, university networks, and social media platforms. Respondents were informed that their participation is voluntary. An electronic consent form preceded the survey, and only consenting participants could proceed. Data were collected anonymously and stored securely to ensure participant confidentiality.     Figure: 1 Overview of Questionnaire Development and Analysis Process   Ethics Approval and Consent to Participate The ethical approval for this study was obtained from the Umm Al-Qura University, Faculty of Medicine, Scientific Research Ethics Committee (HAPO-02-K-012-2024-10-2302). Electronic informed consent was obtained prior to survey completion. Participants were required to read an information page and indicate their consent by selecting the ‘I agree’ option before accessing the questionnaire.   Statistical Analysis Data collection and analysis were conducted using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and IBM SPSS version 29 (IBM Corp., Armonk, NY, USA). Descriptive statistics were generated for all variables, including counts, percentages, means±standard deviations (SD). The total scores for general NO knowledge and specific knowledge about oral bacteria, mouthwash, and NO were tested against different demographic variables using t-tests, ANOVA, and linear regression. P-value of less than 0.05 was considered significant.

A total of 248 dental professionals, including 104 students, participated in the study. The mean age of participants was 28.5±8.1 years. The mean years of professional experience was 3.9±6.2. Participants were recruited from 12 cities across Saudi Arabia including Riyadh, Makkah, Jeddah, Madinah, Taif, Buraidah,Dammam, Khobar, Qatif, Tabuk, Al-Makhwah, and Abha. Participants’ demographic data are summarized in Table 1 Among the participants, 187 (75.4%) reported having heard about NO, while the remaining 61 (24.6%) had not.   Table 1: Demographic Characteristics of the Study Participants Variable Number Percentage Gender Male 68 27.4 Female 180 72.6 Qualification Student 104 41.9 Intern 17 6.9 General Dentistry 42 16.9 Specialist 47 18.9 Consultant 38 15.3 Work Setting Educational (academic) 149 60.1 Private sector 35 14.1 Governmental sector 64 25.2 Saudi Region of Practice Western 139 56.1 Central 78 31.5 Eastern 18 7.3 Southern 5 2.0 Northern 8 3.2   Among participants who reported having heard about NO (n = 187, 75.4%), 57 (30.5%) mistakenly believed that NO is the gas used as local anesthesia (nitrous oxide), and 22 (11.8%) indicated that they were unsure about this distinction. There were only 44 (17.7%) who reported they were familiar with the NO3⁻ – NO2⁻ – NO pathway and its role in human health. Participants answered seven general questions regarding basic information about NO, as shown in Table 2.   Table 2: Participants’ Responses to General Knowledge Questions about NO Question Option Number Percentage In dental practice, NO is used as a local anesthesia Yes 60 24.2 No* 124 50 I do not know 64 25.8 NO is a naturally compound produced in the human body Yes* 78 31.5 No 41 16.5 I do not know 129 52 NO has a significant impact on cardiovascular health Yes* 110 44.4 No 30 12.1 I do not know 108 43.6 NO causes blood vessel constriction Yes 69 27.8 No* 57 22.9 I do not know 122 49.2 NO plays a role in modulating immune response and inflammation Yes* 54 21.8 No 50 20.2 I do not know 144 58.1 The body can produce NO from dietary sources Yes* 72 29 No 32 12.9 I do not know 144 58.1 NO levels in the oral cavity can be affected by periodontal disease Yes* 67 27 No 33 13.3 I do not know 148 59.7 *Indicate the correct answers. NO: Nitric oxide, NO3 – NO2 – NO: Nitrate- Nitrite- Nitric oxide   They also answered four questions regarding specific information about oral bacteria, mouthwash and NO pathway (Table 3).   When participants were asked whether they believed there is a connection between the use of antibacterial mouthwash and the development or progression of cardiovascular diseases, 87 (35.1%) answered "Yes," 41 (16.5%) answered "No," and 120 (48.4%) responded "I do not know." Similarly, regarding whether antibacterial toothpastes have a different effect on NO production compared to antibacterial mouthwash, 58 (23.4%) answered "Yes," 41 (16.5%) answered "No," and 149 (60.1%) responded "I do not know."   Regarding the general knowledge questions about NO, participants achieved a mean score of 2.3±1.9 out of a possible score of 7. Only 17 (6.9%) correctly answered more than 70% of these questions (i.e., more than 5 out of 7). Similarly, for the specific questions related to oral bacteria, mouthwash, and NO, the mean score was 0.9±1.2 out of a possible 4. Only 9 (3.6%) correctly answered more than 70% of these questions (i.e., more than 3 questions out of 4).   Table 3: Participants’ Responses to Specific Questions on Oral Bacteria, Mouthwash Use, and NO Question Option Number Percentage Consuming high amounts of leafy green vegetables and beetroot that are rich in dietary NO3 - can leadto ------ NO levels in the bloodstream Decrease 24 9.7 Increase* 80 32.3 Has no impact on 30 12.1 I do not know 114 45.9 How do presence oral bacteria influence NO levels in the body? Oral bacteria enhance NO levels* 37 14.9 Oral bacteria inhibit NO levels 36 14.5 Oral bacteria have no effect on NO levels 14 5.7 I do not know 161 64.9 The use of antibacterial mouthwash --------------NO production Decrease* 59 23.8 Increase 29 11.7 Has no impact on 18 7.3 I do not know 142 57.3 Which of the following mouthwash has a potent effect on NO production? Chlorhexidine * 53 21.4 Alcohol-based Mouthwash 19 7.7 All types of antibacterial mouthwash equally affect NO production 22 8.9 I do not know 154 62.1 *Indicate the correct answers. CVD: Cardiovascular diseases, NO: Nitric Oxide   Differences in Knowledge between Demographic Groups The total scores for general NO knowledge and specific knowledge regarding oral bacteria, mouthwash, and NO were analyzed in relation to against gender, years of experience, qualification, region, and work setting using simple linear regression, t-tests and ANOVA. None of these demographic variables were found to be statistically significant predictors of knowledge scores (p > 0.05), as shown in Table 4. However, both age and years of practical experience emerged as significant predictors of general NO knowledge. Age had a small but statistically significant effect (β = 0.128, p = 0.043, R² = 0.016), and years of practical experience also showed a significant association (β = 0.146, p = 0.021, R² = 0.021). In contrast, neither age (p = 0.955) nor years of practical experience (p = .700) were significantly associated with specific knowledge as shown in Table 4. Additionally, participants who had previously heard of NO scored significantly higher in both general NO knowledge and specific knowledge about oral bacteria, mouthwash, and NO compared to those who had not (p<0.001for both comparisons, t-test), as detailed in Table 4.   Table 4: Demographic Variables, against General Knowledge, and Specific Knowledge Variable General Knowledge Specific Knowledge Mean SD P-value Mean SD p-value Age* - - 0.04 - - 0.96 Years of experience* - - 0.02 - - 0.70 Gender** Male 2.5 1.9 0.27 0.8 1.1 0.32 Female 2.2 1.9 0.9 1.2 Qualification*** Student/ intern 2.1 1.9 0.28 0.9 1.2 0.98 Working dentist 2.4 1.9 0.9 1.2 Setting of work*** Educational (academic) 2.2 1.7 0.72 0.9 1.2 0.34 Private sector 2.2 2.0 1.1 1.1 Governmental sector 2.5 2.0 0.9 1.2 Region*** Western 2.4 1.9 0.52 1.0 1.3 0.67 Central 2.1 2.0 0.8 1.1 Western 2.0 1.6 0.7 1.0 Southern 2.4 1.1 0.2 0.5 Northern 2.6 1.7 1.1 1.3 Have you heard about Yes 2.7 1.9 <0.001 1.1 1.2 <0.001 nitric oxide (NO)?** No 1.0 1.5 0.5 1.0 *Linear regression, **t-test, ***ANOVA

This study evaluated the knowledge of Saudi dental professionals and students regarding the NO pathway, the role of oral bacteria, and the impact of antiseptic mouthwash use on NO production. It also compared knowledge levels across different demographic and professional groups. A key finding was that three-quarters of participants reported having heard about NO; however, nearly one-third mistakenly believed that NO is the same as nitrous oxide, the gas commonly used for local anesthesia. This highlights a significant misconception among dental professionals regarding the basic identity and function of NO. Alternatively, these responses could suggest a lack of careful attention while completing the questionnaire. Overall, participants showed limited knowledge of both general concepts related to NO and specific information concerning oral bacteria and the effects of mouthwash on NO production. These results highlight notable gaps in knowledge among Saudi dental professionals in an area directly relevant to oral and systemic health.   NO is a key signaling molecule that is produced endogenously in the human body via an enzymatic pathway which depends on NOS and the substrate L-arginine [16]. NO has a well-established role in vascular homeostasis including vasodilation [25], regulation of blood pressure [26] and overall cardiovascular health. It also contributes to host defense by modulating inflammation and immune responses [27]. Despite the significance of NO in maintaining systemic health, our findings revealed considerable knowledge gaps among dental professionals in these aspects. Less than one-third of our participants were aware that NO is naturally synthesized in the body, and less than half of them identified its importance in cardiovascular health. However, a substantial portion of respondents were uncertain about the vasodilatory effect of NO (49%), with only 27.8% identifying this function correctly. Knowledge of NO’s role in modulating immune response and inflammation was even more limited, with the majority of participants lacking clarity on this relationship. This illustrates the limited understanding of the systemic functions of NO among dental professionals. These findings suggest that the limited knowledge observed may be attributed to gaps in dental education, insufficient interdisciplinary training, and a lack of emphasis on NO's systemic roles in professional development resources.   Dietary NO3⁻, primarily from vegetables such as green leafy vegetables and beetroot, is considered a secondary but essential source of NO in the body through entero-salivary circulation pathway [16]. However, our findings indicate that this concept is not well understood among dental professionals as the majority of respondents expressed uncertainty about the nutritional contribution to NO production. This limited understanding was further reflected in responses to a related item in the specific knowledge section, where only about one third of respondents recognized that consuming NO3⁻-rich foods such as leafy greens and beetroot can increase NO levels in the bloodstream. Nearly half of the respondents were uncertain about the role of dietary NO₃⁻ in enhancing NO levels through consumption of foods like leafy greens and beetroot. While it may be understandable that dental professionals are less familiar with the nutritional aspects of NO metabolism, these findings highlight an important interdisciplinary gap. Given that NO3⁻-reducing oral bacteria play a key role in converting dietary NO3⁻to NO, a process that can be disrupted by antiseptic mouthwash, thus basic understanding of this pathway is becoming increasingly relevant to dental practice. This gap was clearly demonstrated in our results; when participants were asked about the role of oral bacteria in influencing NO levels, only 14.9% of participants correctly recognized that oral bacteria enhance NO production, while nearly two-thirds indicated that they did not know. In addition, over a half of our participants were uncertain if using antiseptic mouthwash can affect NO production.   Studies reported that antiseptic mouthwash, particularly chlorhexidine mouthwash, can substantially affecting dietary NO₃⁻metabolism. Woessner et al, assessed the effect of different strengths of mouthwash including chlorhexidine, Listerine antiseptic mouthwash and Cepacol antibacterial mouthwash against control water, on salivary and plasma NO₃⁻and NO2⁻concentrations, after ingestion of beetroot juice (a common NO₃⁻ donor). Chlorhexidine appeared to have the strongest effect on elimination any conversion of salivary NO₃⁻ to NO2⁻, and subsequently reduced NO production [28]. In our study, the majority of participants were unsure about which mouthwash has the greatest impact on NO production, with only about one-fifth correctly identifying chlorhexidine as the most potent inhibitor. In addition, several studies showed that using Chlorhexidine abolished the blood pressure-lowering benefits of dietary NO₃⁻, with some studies reporting increases of 3.5 mmHg or more in healthy individuals [29-31]. A recent meta-analysis found that regular use of antiseptic mouthwash is associated with a slightly but significantly increased risk of developing hypertension [32]. Furthermore, some epidemiological studies reported that there is also an association between regular use of antiseptic mouthwash and increase the risk of diabetes [14]. This growing evidence was not reflected in participants responses with half of them stating that they are unsure if there is a link between antibacterial mouthwash use and the development or progression of future cardiovascular disease. The lack of such knowledge may lead to underestimating the broader health implications of mouthwash use beyond oral hygiene, particularly its potential impact on vascular and metabolic functions. Recommending frequent antiseptic mouthwash use without consideration of such systemic effects could unintentionally contribute to negative health outcomes, especially in vulnerable populations.   While the potential systemic effects of antiseptic mouthwash on NO production are becoming more evident, less is known about the role of antibacterial toothpastes. When participants were asked whether antibacterial toothpastes might affect NO production similarly to mouthwash, most of them were uncertain. In 2012, Bondonno and colleagues examined the effect of antibacterial toothpastes on NO₃⁻ reduction and circulating NO, and they found that it did not affect either of them [33]. The authors explained that triclosan, which is a common antibacterial ingredient in toothpaste, is unable to effectively penetrate the biofilm within deep clefts where oral NO₃⁻ -reducing bacteria are primarily located [33]. While descriptive analysis indicated no significant differences in knowledge across demographic groups, regression analysis revealed that both age and years of practical experience were significant predictors of general knowledge, though the effect sizes were modest (age: β = .128, p = 0.04; experience β = 0.146, p = 0.02). In contrast, specific knowledge related to the NO₃⁻-NO2 –NO pathway, oral bacteria, and mouthwash use did not differ significantly by age (p = 0.96) or experience (p = 0.700). These findings suggest that while overall awareness may slightly improve with professional experience, detailed understanding remains consistently low across the sample.   This study provides novel insights into knowledge of the dietary NO₃⁻-NO2 –NO pathway and the impact of mouthwash use on it among dental professionals in Saudi Arabia, via using a newly developed questionnaire. This tool may also be applicable for assessing similar knowledge among dental professionals in other countries, tracking knowledge in the same group of individuals over time, or within the general population. On the other hand, our study has certain limitations. The sample size was relatively small and self-selected, which may limit the generalizability of the results to the larger population of dental professionals in Saudi Arabia. Although efforts were made to reach a large audience by distributing the questionnaire across various professional networks, dental institutions, and regions, it is possible that not all relevant subgroups within the dental community were adequately represented. In addition, our study aimed to include participants from various regions of Saudi Arabia, the distribution of the responses though was uneven, with most of them coming from western and central regions. This likely reflects the recruitment strategy which relied on institutional and community networks primarily based in the Western region. Although this may limit the representativeness, the data still shows valuable insights into awareness among dental professionals from different cities in Saudi Arabia. Another limitation was that we administered a relatively small number of closed-ended questions. This was a deliberate design feature to maximize response rate. Future follow-up qualitative research incorporating open-ended questions or interviews could be valuable in the future to assess the depth of participants’ understanding. Finally, it cannot be ruled out that the low level of knowledge displayed in some individuals could reflect a lack of attention (e.g., mistaking NO for nitrous oxide), rather than a genuine lack of knowledge.

This study provides novel insights into general knowledge of NO and understanding of impact of oral bacteria and antiseptic mouthwash on NO production among Saudi dental professionals. Our findings highlight significant gaps in understanding the physiological role of NO and how oral care practices may influence its availability. Despite the growing body of evidence linking the oral microbiome, antiseptic mouthwash and dietary NO₃⁻ to systemic health outcomes, many participants were unaware of the role of oral bacteria and antiseptic mouthwash in NO₃⁻ reduction and NO production. Notably, confusion between NO and nitrous oxide was also apparent. These results underscore the need to enhance dental education by integrating updated knowledge about NO, its dietary sources, and the broader connection between oral and systemic health. Given the observed gaps in knowledge regarding NO functions and its derivatives, we recommend integrating more comprehensive content related to NO and its precursors into dental education and continuing professional development programs. Emphasizing the role of NO₃⁻ and NO in cardiovascular health, immune modulation, and inflammatory responses may enhance interdisciplinary understanding and promote more holistic patient care among dental professionals. Evidence-based guidelines on the use of antiseptic mouthwashes should be developed, balancing oral health benefits with potential systemic risks, especially in patients with cardiovascular or metabolic vulnerabilities.