Cytotoxicity, Genotoxicity and Cytokines Modulation of N-acetylcysteine, Calcium Hydroxide, Chlorhexidine and their Combinations

Cytotoxicity, Genotoxicity and Cytokines Modulation of N-acetylcysteine, Calcium Hydroxide, Chlorhexidine and their Combinations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cytotoxicity, Genotoxicity and Cytokines Modulation of N-acetylcysteine, Calcium Hydroxide, Chlorhexidine and their Combinations Ana Caroline Freitas Da Silva, Sabline Martinele Soares Silva, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9186924/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to evaluate the cytotoxicity, genotoxicity and effect on cytokine production of n-acetylcysteine (NAC), calcium hydroxide [Ca(OH)₂], chlorhexidine (CHX) and their combinations on murine macrophage cells (RAW 264.7) and human osteoblasts-like cells (MG-63). Cytotoxicity was evaluated by MTT colorimetric assay after 5 min and 24 h. Genotoxicity evaluation was evaluated by cytokinesis-block micronucleus assay. In addition, cytometry bead arrays to quantify different cytokine production by RAW 264.7 and MG63 cells. Data were statistically analyzed with a significant level of α ≤ 0.05. All medications promoted high cellular viability of RAW 264.7 after 5 min, and of MG63 after 24h. All medications presented a low number of micronuclei except for CHX in Raw 264.7. NAC was highlighted as it stimulates high production of IL-12p70, IL-6, and IFN-γ in RAW 264.7 cells, in addition to IL-12p70, TNF-α, IL-10, IL-6, and IL-1β in MG63. Therefore, NAC demonstrated a potential immunomodulatory effect by promoting a balance between pro-inflammatory activation and regulatory cytokine responses. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Immunology Health sciences/Medical research acetylcysteine calcium hydroxide chlorhexidine Cytotoxicity Genotoxicity Cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Endodontic infections are characterized by a complex polymicrobial etiology in which diverse bacterial species and their associated pathogen-associated molecular patterns (PAMPs), including lipoteichoic acid (LTA) and lipopolysaccharide (LPS), play a critical role [ 1 ] . The presence of microorganisms and their metabolic by-products activates the host immune response, initiating an inflammatory cascade primarily mediated by the innate immune system. This early response involves neutrophils, macrophages, dendritic cells, and activation of the complement system, and is characterized by phagocytosis and the release of pro-inflammatory cytokines and chemokines. However, persistent microbial invasion subsequently promotes the activation of the adaptive immune response, characterized by the recruitment and activation of T and B lymphocytes to enhance antigen-specific immune regulation and contribute to the containment and modulation of the infection [ 2 – 4 ] . A complex cytokine network is involved to regulate the inflammatory response including pro-inflammatory cytokines like interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) which play critical roles in promoting osteoclast differentiation and activation through upregulation of RANKL expression [ 5 , 6 ] . IL-8 functions as a potent neutrophil chemoattractant, contributing to the maintenance of the acute inflammatory infiltrate, whereas IL-12 supports T-helper 1 (Th1) polarization and sustains a cell-mediated inflammatory response [ 7 – 9 ] . Chemokines such as monocyte chemoattractant protein-1 (MCP-1) further regulate recruitment of monocytes and macrophages, reinforcing the chronic inflammatory environment [ 10 , 11 ] . Additionally, interferon-gamma (IFN-γ) contributes to macrophage polarization and perpetuation of the pro-inflammatory phenotype [ 12 ] . Conversely, regulatory cytokines such as interleukin-10 (IL-10) act to suppress excessive production of pro-inflammatory mediators, limit osteoclastogenic signaling, and contribute to immune balance and tissue repair [ 13 ] . The dynamic interplay between pro-inflammatory and regulatory mediators ultimately determines whether periapical lesions progress or shift toward resolution, influenced by bacterial virulence, host immune regulation and the efficacy of the treatment. Root canal treatment, in turn, is indicated to combat the invasion of the bacteria and their by-products, and to control the inflammatory process using files, endodontic irrigants and eventually intracanal medications like calcium hydroxide [Ca(OH)₂], which is commonly used because of its antimicrobial action, ability to neutralize endotoxins, ability to induce tissue repair through the deposition of mineralized tissue, and having a good tolerance by periapical tissues [ 14 ] . Ca(OH)₂ can reduce the production of pro-inflammatory cytokines, such as IL-1β and IL-6, and has the capacity to neutralize the cytotoxic effects of LPS and LTA [ 15 , 16 ] . Besides to, chlorhexidine digluconate (CHX) which is also widely used intracanal medication because of its biocompatibility and broad spectrum of antimicrobial activity [ 17 ] , besides to its efficacy in reducing different pro-inflammatory cytokines [ 15 , 16 ] N-acetylcysteine (NAC) is a derivative of L-cysteine, with recognized mucolytic and antioxidant action, acting as a precursor of glutathione and contributing to the neutralization of free radicals. Although it is not an antibiotic medication, it presents antimicrobial properties and the ability to reduce biofilm formation by inhibiting the production of extracellular polysaccharides, in addition to promoting the disruption of mature biofilms and decreasing bacterial adhesion [ 18 – 20 ] . NAC also presents an inhibitory effect on the expression of several pro-inflammatory cytokines [ 21 ] increases the levels of Resolvin [ 22 ] , and has been indicated as a promising intracanal medication, as it demonstrates activity against relevant endodontic pathogens [ 23 , 24 ] To the best of our knowledge, NAC, Ca(OH)₂, CHX, and their combinations have not yet been evaluated regarding their cytotoxicity, genotoxicity and effects on cytokine production. Therefore, the aim of this study was to assess the cytotoxicity, genotoxicity, and anti-inflammatory potential of these intracanal medications, alone, and in combination. The null hypothesis tested was that these medications would not be biocompatible and would not exert any anti-inflammatory effect. MATERIAL AND METHODS Sample size calculation It was performed using the online calculator: http://estatistica.bauru.usp.br/calculoamostral/calculos.php with the following parameters: 8 groups, an estimated standard deviation of 2, a minimum detectable difference of 3.6, α = 5%, and β = 20% (80% power). The minimum required sample size was determined to be 10 per group (n = 10). Experimental groups Control group: saline solution (Eurofarma, São Paulo, SP, Brazil); Ca(OH)₂: The Ca(OH)₂ powder (Biodinâmica Química e Farmacêutica LTDA, Paraná, Brazil) was with sterile saline solution (1:1 ratio, 100 µg of powder and 100 µL of sterile saline solution). CHX: 100 µL of 2% chlorhexidine gel (Terapêutica Farmácia de Manipulação, São José dos Campos, SP, Brazil); NAC: The NAC powder (Merck KGaA, Darmstadt, Germany) was mixed with sterile saline solution (1:1 ratio, 100 µg of powder and 100 µL of sterile saline solution). NAC + Ca(OH)₂: The NAC and Ca(OH)₂ powders was mixed with sterile saline solution (0.5:0.5:1 ratio, 50 µg of NAC powder + 50 µg of Ca(OH)₂ powder and 100 µL of sterile saline solution). NAC + CHX: The NAC powder was mixed with CHX gel (1:1 ratio, 100 µg of powder and 100 µL of CHX gel) Ca(OH)₂ + CHX: The Ca(OH)₂ powder was mixed with CHX gel (1:1 ratio, 100 µg of powder and 100 µL of CHX gel). NAC + Ca(OH)₂ + CHX: The NAC and Ca(OH)₂ powders was mixed with CHX gel (0.5:0.5:1 ratio, 50 µg of NAC powder + 50 µg of Ca(OH)₂ powder and 100 µL of CHX gel). In a 24-well plate, the medications were prepared according to the previously established proportion. Each well was filled with 2 mL of Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, New York, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% amphotericin B, and kept in an incubator with 5% CO₂ at 37°C for 24 h. Later, 100 µL of the conditioned medium previously in contact with each medication was used as a treatment and transferred to the corresponding wells containing cultured cells according to the respective experimental group. Cell Culture Murine macrophage cells (RAW 264.7) and human osteoblasts-like cells (MG-63) were used, obtained from the Cell Bank of Rio de Janeiro (APABCAM, RJ, Brazil). The cells were cultured in supplemented DMEM and maintained in an incubator at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was replaced every 48 h until the required number of cells was obtained. For the experiments, RAW 264.7 cells were mechanically detached from the bottom of the flasks using a cell scraper, whereas MG-63 cells were chemically detached with 0.25% trypsin-EDTA solution (Cultilab, Campinas, SP, Brazil). The quantification of viable cells was performed using the Trypan blue exclusion test (0.4%, Sigma-Aldrich, St. Louis, MO, USA). Cytotoxicity evaluation – MTT colorimetric assay The viable cells (2 × 10⁴) were cultivated in 96-well microplates at 37°C with 5% CO₂ for 24 h to allow cell adhesion. After incubation, the culture medium was removed, and the wells were gently washed with sterile phosphate-buffered saline (PBS) (Cultilab, Campinas, SP, Brazil). The cells were treated with 100 µL of the conditioned medium of the respective intracanal medication for 5 mins or 24 h being incubated under the same conditions (37°C, 5% CO₂). Later, cell viability was determined by assessing mitochondrial metabolic activity through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystals by viable cells. The MTT solution was prepared by dissolving 0.5 mg of MTT powder (Sigma-Aldrich, Germany) in 1 mL of sterile PBS. Subsequently, 100 µL of MTT solution was added to each well, and the plates were incubated for 4 h at 37°C in a humidified atmosphere containing 5% CO₂, protected from light. After incubation, the MTT solution was carefully discarded, and 100 µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well to solubilize the formazan crystals formed by viable cells. The plates were incubated for 10 min and gently shaken for an additional 10 min to ensure complete dissolution. The absorbance was measured at 570 nm using a microplate reader. Optical density (OD) values were converted into percentage of cell viability using the following formula: $$\:Cell\:viability\:=\frac{\:OD\:of\:treated\:group\:\times\:\:100}{Mean\:of\:OD\:of\:control\:group}$$ Genotoxicity evaluation - cytokinesis-block micronucleus assay Differently, the viable cells were seeded in 24-well plates at a density of 5 × 10⁵ cells/mL and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. The cells were treated with 100 µL of the conditioned medium of the respective intracanal medication for 24 h being incubated under the same conditions (37°C, 5% CO₂). Ethyl methane sulfonate (EMS) was used as a control group because of its capacity in forming micronuclei. Subsequently, cytochalasin B (Sigma-Aldrich) was added at a final concentration of 6 µg/mL, and the cells were incubated for an additional 24 h at 37°C with 5% CO₂ to inhibit cytokinesis and allow the formation of binucleated cells. Then, the cells were subjected to hypotonic treatment and fixed with a methanol:acetic acid solution (3:1) for 10 minutes. The fixation procedure was repeated three times. The wells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, Missouri, USA), and the dye was removed after 5 min of contact with the cells and then washed with PBS. Micronucleus analysis was performed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) at 400× magnification. A total of 2,000 cells per well were evaluated in at least two independent experiments. Micronuclei were identified as small DNA-containing structures located in the cytoplasm, clearly separated from the main nucleus, surrounded by a nuclear membrane, and with an area smaller than one-third of the main nucleus. Only cells containing fewer than five micronuclei were included in the analysis. Anti-inflammatory evaluation - Cytometric Bead Array The viable cells were seeded into 24-well plates at a density of 1 × 10⁵ cells per well and incubated for 24 h to allow cell adhesion. The cells were treated with 100 µL of the conditioned medium of the respective intracanal medication for 24 h. Simultaneously, LPS of Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA) was added to each well at a final concentration of 5 µg/mL to induce an inflammatory response. The plates were then incubated for 24 h at 37°C in a humidified atmosphere with 5% CO₂. Later, the supernatants were collected and stored at − 20°C until cytokine quantification. For RAW 264.7 cells, IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70 were quantified using the Mouse Inflammation Kit (BD Biosciences). For MG-63 cells, cytokine levels (IL-8, IL-1β, IL-6, IL-10, TNF, and IL-12p70) were measured using the Human Inflammatory Cytokine Kit (BD Biosciences, San Diego, CA, USA). Cytokine analysis for both cell lines was performed by flow cytometry using the Cytometric Bead Array (CBA) system, according to the manufacturer’s instructions. Statistical analysis Data were tabulated and submitted to Shapiro–Wilk normality test. Then, non-parametric data were analyzed by Kruskal Wallis and complemented by Dunn for multiple comparisons. Whereas parametric data were analyzed by one-way Anova and complemented by Tukey for multiple comparisons. All these tests were performed considering a significant level of α ≤ 0.05 using GraphPad Prism 10.6.1 (GraphPad Software, San Diego, CA, USA). RESULTS Cytotoxicity evaluation After 5 min of treatment, all the experimental groups presented cellular viability of RAW 264.7 cells without a statistically significant difference with the control group except of Ca(OH) 2 , CHX, NAC + Ca(OH) 2 , and NAC + Ca(OH) 2 + CHX groups that promoted the cellular viability more than saline solution control group with a statistically significant difference (P < 0.0001) up to 92.4, 263.53, 92.67 and 252.38%, respectively. Nevertheless, after 24 h of treatment, all the experimental groups presented cellular viability without a statistically significant difference with the control group except of NAC, NAC + CHX and Ca(OH) 2 + CHX groups that presented lower values of viable cells (-19.1, -14.42 and − 19.12, respectively) with a statistically significant difference (P < 0.0001) (Fig. 1 ). In contrast, after 5 min and 24 h of treatment, all the experimental groups presented cellular viability of MG63 cells without a statistically significant difference with the control group (P > 0.05) except of NAC group after 5 min that reduced the cellular viability to − 14.32% and Ca (OH) 2 + CHX after 24 h that promoted the cellular viability with 35.20% with a statistically significant difference with the control group (Fig. 1 ). Genotoxicity evaluation After 24 h of treatment, all the experimental groups presented a relatively lower number of micronuclei in RAW 264.7 and MG63 cells in comparison with the EMS group with a statistically significant difference (P < 0.0001). Except of CHX, which presented a high number of micronuclei in RAW 264.7 cells being as genotoxic as EMS statistically as seen in Fig. 2 . Anti-inflammatory evaluation In RAW 264.7, NAC group induced the production of high values of IL-12p70 with a statistically significant difference (P < 0.0001) with all the experimental groups, of IL-10 with a statistically significant difference (P < 0.0001) with all the experimental groups except of NAC + CA(OH) 2 and NAC + CHX groups, and of IFN-γ with a statistically significant difference (P < 0.0001) with all the experimental groups except of NAC + CHX group. Differently, Ca(OH) 2 group induced the production of high values of TNF with a statistically significant difference (P < 0.0001) with all the experimental groups except of NAC and Ca(OH) 2 + CHX group, and of MCP-1 with a statistically significant difference (P < 0.0001) with all the experimental groups. Nevertheless, NAC, Ca(OH) 2 and NAC + CHX groups induced the production of high values IL-6 with a statistically significant difference (P < 0.0001) as seen in Fig. 3 . In MG63, NAC induced the production of high values IL-12p70, TNF, IL-10 and IL-1β, with a statistically significant difference (P < 0.0001) with all the experimental groups. In addition, NAC and Ca(OH) 2 + CHX group induced the production of high values IL-6 with a statistically significant difference (P < 0.0001) in comparison with all the experimental groups except of CHX which was statistically similar. Differently, Ca(OH) 2 , CHX, Ca(OH) 2 + CHX groups induced the production of high values IL-8 with a statistically significant difference (P < 0.0001) in comparison with all the experimental groups as seen in Fig. 4 . DISCUSSION The present study evaluated the cytotoxicity, genotoxicity, and inflammatory response–modulating potential of NAC, Ca(OH)₂, CHX and their combinations on RAW 264.7 macrophages and MG-63 osteoblast-like cells. Overall, the results showed that some medications demonstrated acceptable biocompatibility, whereas others exhibited potential genotoxic effects or modulated the production of pro- and anti-inflammatory cytokines. Therefore, the null hypothesis was partially rejected. In the present study, all tested intracanal medications demonstrated high cellular viability of MG63 after treatment for 5 min and 24 h being as biocompatible as saline solution except of NAC after 5 min that reduced the cellular viability by 14.32%. Although NAC slightly reduced the cellular viability of MG63 at the 5-minute time point, cell viability after 24 h was comparable to that observed with the other tested medicaments. In the literature, NAC was added to zinc oxide–eugenol-based materials to improve their biocompatibility in MG63 [ 25 ] . Otherwise, no significant reductions in cell viability were observed. This align with the results in the literature that confirm the biocompatibility of Ca(OH) 2 , CHX and their combination in osteoblasts [ 26 ] T This suggests that, under the experimental conditions of the present study, the tested materials did not directly compromise the metabolic activity of cells involved in bone repair processes. From a biological perspective, this observation is particularly relevant, as osteoblasts play a central role in bone formation, resolution of periapical lesions, and modulation of the local inflammatory microenvironment [ 27 ] Therefore, the maintenance of MG-63 cell viability indicates that the tested medications did not significantly interfere with the basal metabolic activity of these cells, thereby preserving their potential to contribute to tissue repair. In contrast, RAW 264.7 cells exhibited a response that was dependent on exposure time. After 5 minutes, all experimental groups showed high biocompatibility, in some cases exceeding that observed in the saline solution, particularly in the Ca(OH)₂, CHX, NAC + Ca(OH)₂, and NAC + Ca(OH)₂ + CHX groups. As highly adaptable cells, macrophages are capable of rapidly adjusting their energy metabolism in response to chemical and inflammatory stimuli [ 28 ] . Considering that the MTT assay evaluates the activity of the mitochondrial enzyme succinate dehydrogenase, alterations in cellular metabolic state may directly influence the observed viability indices [ 29 ] The reduction in viability observed after 24 h in the NAC, NAC + CHX, and Ca(OH)₂ + CHX groups may be associated with redox imbalance or prolonged metabolic stress. CHX has been reported to induce mitochondrial dysfunction and alterations in cell membrane integrity in a manner dependent on concentration and exposure time [ 17 ] . Although NAC is widely recognized for its antioxidant properties, it may also modulate signaling pathways involving reactive oxygen species (ROS) at certain concentrations, potentially affecting cellular dynamics [ 30 ] . Additionally, the Ca(OH)₂ + CHX association deserves attention. While Ca(OH)₂ is generally considered biocompatible and capable of inducing mineralization, its high pH may cause transient alterations in cellular homeostasis and may also affect the chemical stability of CHX in highly alkaline environments [ 31 ] . Regarding genotoxicity, the results revealed distinct behaviors among the tested medications. A significant increase in micronucleus number was observed in the groups containing CHX, particularly in RAW 264.7, whereas NAC and Ca(OH)₂ did not promote relevant genomic damage under the experimental conditions, however, all tested medications presented a relatively low number of micronuclei in MG63. The presence of micronuclei indicates chromosomal breaks or errors in chromosome segregation during mitosis; events often associated with oxidative stress. Under such conditions, ROS can induce oxidative lesions that, if not properly repaired, may compromise cellular genomic stability [ 32 ] . Previous studies have reported that CHX can induce oxidative stress and DNA damage in a dose-dependent manner, increasing ROS production, and activating apoptotic pathways [ 33 ] . These mechanisms are consistent with the increased micronucleus frequency observed in the present study, particularly in inflammatory cells, which typically exhibit higher basal ROS production. In contrast, although Ca(OH)₂ presents a highly alkaline pH, it did not significantly increase micronucleus formation under the experimental conditions, suggesting that its alkalinity alone was not genotoxic in this model, this was affirmed over stem cells of apical papilla in a previous study [ 34 ] . Furthermore, NAC demonstrated a stable biological profile as it can reduce oxidative DNA damage and improving cellular resistance to genotoxic stress by scavenging ROS and supporting intracellular glutathione levels [ 35 ] In this study, RAW 264.7 and MG63 cells were stimulated with E. coli LPS to mimic an inflammatory microenvironment. Therefore, cytokine production should be interpreted in the context of LPS-induced cells activation and the modulatory effects of the tested medications [ 36 ] . Under these conditions, NAC increased the production of the pro-inflammatory cytokines IL-12p70, IL-6, and IFN-γ in RAW 264.7 cells. This response may be associated with the reduction in RAW 264.7 viability observed after 24 h in the NAC group, as decreased viability may reflect a state of metabolic or redox stress [ 37 ] . In such conditions, macrophages can activate inflammatory signaling pathways that contribute to increased cytokine production. Interestingly, NAC also increased the production of IL-10, an anti-inflammatory cytokine, suggesting that NAC may exert an immunomodulatory effect by balancing inflammatory activation and regulatory responses. In vitro study confirmed that NAC can adjust the production of mediators such as TNF-α, IL-1β, IL-6, and IL-8 in activated macrophages, refining the final inflammatory response [ 38 ] . Differently, Ca(OH)₂ induced the production of only pro-inflammatory cytokines, including TNF-α, IL-6, and MCP-1. These findings are consistent with a previous study reporting that Ca(OH)₂ stimulates the production of TNF-α in macrophages exposed to LTA from Enterococcus faecalis [ 15 ] . Furthermore, the same study demonstrated that the combination of Ca(OH)₂ and CHX also promoted TNF-α production, which agrees with the results observed in the present study. This effect may be related to the high alkalinity of Ca(OH)₂, which can activate macrophages and influence inflammatory signaling pathways. Another finding that deserves attention is the cytokine profile observed in the NAC + CHX group, which showed increased production of IL-10, IL-6, and IFN-γ. This pattern suggests that the addition of NAC to CHX may exert a modulatory effect on the inflammatory response, potentially contributing to a more balanced regulation between pro- and anti-inflammatory signaling. In MG-63 cells, a consistent increase in the production of IL-12p70, TNF-α, IL-10, IL-6, and IL-1β was observed following treatment with NAC compared with the other experimental groups. This finding suggests that NAC directly influenced the functional activity of these osteoblast-like cells while preserving their potential for tissue repair [ 39 ] . The concomitant increase in IL-10 indicates that the response induced by NAC was not exclusively pro-inflammatory but also involved regulatory mechanisms controlling the inflammatory response. IL-10 is recognized as an important anti-inflammatory mediator, as it can limit the production of pro-inflammatory cytokines and prevent excessive tissue damage [ 40 ] . Considering the ability of NAC to modulate intracellular redox status, the observed changes in cytokine production may be associated with alterations in ROS-dependent signaling pathways. Such modulation may influence cellular processes involved in periapical bone repair, highlighting the potential relevance of NAC in regulating the inflammatory microenvironment associated with periapical tissues. The combinations of the tested medications NAC + Ca(OH) 2 , NAC + CHX, Ca(OH) 2 +CHX and NAC + Ca(OH) 2 +CHX generally did not produce marked alterations in the levels of pro- or anti-inflammatory cytokines, except for a few isolated findings. Overall, these combinations also demonstrated acceptable biocompatibility and did not significantly increase micronucleus formation. CONCLUSIONS The tested medications maintained high cellular viability of RAW 264.7 cells after 5 min of exposure and MG-63 cells after 24 h. The medications exhibited low genotoxic potential, with a low frequency of micronuclei formation in most groups, except for CHX in RAW 264.7 cells. NAC demonstrated a potential immunomodulatory effect by promoting a balance between pro-inflammatory activation and regulatory cytokine responses. Declarations Competing interests: The authors deny any conflicts of interest related to this study. Funding: This work was financially supported by São Paulo Research Foundation (FAPESP) 2023/10158-2 and by the Institutional Scholarship Program for Scientific Initiation (PIBIC) of the National Council for Scientific and Technological Development (CNPq). Author Contribution Conceptualization: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., T.A-N., L.D.O., and A.A.H.; Data curation: A.C.F.S., S.M.S.S., L.D.O., and A.A.H.; Formal analysis: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Funding acquisition: V.A.M., and A.A.H.; Investigation: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Methodology: A.C.F.S., S.M.S.S., T.A-N., L.D.O., and A.A.H.; Project administration: A.A.H.; Resources: L.D.O., and A.A.H.; Software: L.D.O., and A.A.H.; Supervision: T.A-N., L.D.O., and A.A.H.; Validation: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Visualization: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Roles/Writing - original draft: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., and J.S.L.; and Writing - review & editing: T.A-N., L.D.O., and A.A.H. Acknowledgement None. 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Antimicrobial and anti-endotoxin activity of N-acetylcysteine, calcium hydroxide and their combination against Enterococcus faecalis, Escherichia coli and lipopolysaccharides. PeerJ 12, e18331 (2024). Chang, M. C. et al. Toxic mechanisms of Roth801, Canals, microparticles and nanoparticles of ZnO on MG-63 osteoblasts. Mater. Sci. Eng. C Mater. Biol. Appl. 119 , 111635 (2021). Sy, K. et al. Therapeutic Potential of Chlorhexidine-Loaded Calcium Hydroxide-Based Intracanal Medications in Endo-Periodontal Lesions: An Ex Vivo and In Vitro Study. Antibiotics (Basel) 12 , (2023). Wen, Y. H., Lin, Y. X., Zhou, L., Lin, C. & Zhang, L. The immune landscape in apical periodontitis: From mechanism to therapy. Int. Endod J. 57 , 1526–1545 (2024). O’Neill, L. A. J. & Pearce, E. J. Immunometabolism governs dendritic cell and macrophage function. J. Exp. Med. 213 , 15–23 (2016). Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods . 65 , 55–63 (1983). Kalyanaraman, B. & NAC, N. A. C. Knockin’ on Heaven’s door: Interpreting the mechanism of action of N-acetylcysteine in tumor and immune cells. Redox Biol. 57 , 102497 (2022). Mohammadi, Z. & Abbott, P. V. The properties and applications of chlorhexidine in endodontics. Int. Endod J. 42 , 288–302 (2009). Fenech, M. The in vitro micronucleus technique. Mutat. Research/Fundamental Mol. Mech. Mutagen. 455 , 81–95 (2000). Li, Y. C., Kuan, Y. H., Lee, S. S., Huang, F. M. & Chang, Y. C. Cytotoxicity and genotoxicity of chlorhexidine on macrophages in vitro. Environ. Toxicol. 29 , 452–458 (2014). Jamshidi, D., Ansari, M. & Gheibi, N. Cytotoxicity and genotoxicity of calcium hydroxide and two antibiotic pastes on human stem cells of the apical papilla. Eur. Endod J. 6 , 303–308 (2021). Yang, Q., Shi, L., Huang, K. & Xu, W. Protective effect of N-acetylcysteine against DNA damage and S-phase arrest induced by ochratoxin A in human embryonic kidney cells (HEK-293). Food Chem. Toxicol. 70 , 40–47 (2014). Lan, C. et al. Different expression patterns of inflammatory cytokines induced by lipopolysaccharides from Escherichia coli or Porphyromonas gingivalis in human dental pulp stem cells. BMC Oral Health . 22 , 121 (2022). Brüne, B. et al. Redox control of inflammation in macrophages. Antioxid. Redox Signal. 19 , 595–637 (2013). Faghfouri, A. H. et al. The effects of N-acetylcysteine on inflammatory and oxidative stress biomarkers: A systematic review and meta-analysis of controlled clinical trials. Eur. J. Pharmacol. 884 , 173368 (2020). Xu, J., Yu, L., Liu, F., Wan, L. & Deng, Z. The effect of cytokines on osteoblasts and osteoclasts in bone remodeling in osteoporosis: a review. Front. Immunol. 14 , 1222129 (2023). Acuner-Ozbabacan, E. S. et al. The structural network of Interleukin-10 and its implications in inflammation and cancer. BMC Genom. 15 (Suppl 4), S2 (2014). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9186924","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":615231475,"identity":"dacd4f1b-a5d2-424f-8dd2-0b24075b1046","order_by":0,"name":"Ana Caroline Freitas Da Silva","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Caroline Freitas Da","lastName":"Silva","suffix":""},{"id":615231476,"identity":"a0434286-a339-43e9-a584-b6fbbe44d489","order_by":1,"name":"Sabline Martinele Soares Silva","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Sabline","middleName":"Martinele Soares","lastName":"Silva","suffix":""},{"id":615231477,"identity":"574e420f-a1f3-4801-9643-a2f699659748","order_by":2,"name":"Dyovana Ribeiro","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Dyovana","middleName":"","lastName":"Ribeiro","suffix":""},{"id":615231478,"identity":"7e0575a7-d233-4dca-81af-a49c4345c433","order_by":3,"name":"Victória Aparecida Morais","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Victória","middleName":"Aparecida","lastName":"Morais","suffix":""},{"id":615231479,"identity":"56f9fd25-17b1-4652-aa32-551b11ceed61","order_by":4,"name":"Lara Steffany de Carvalho","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"Steffany","lastName":"de Carvalho","suffix":""},{"id":615231480,"identity":"b40a13e2-d607-43e1-80f8-a667b0d90191","order_by":5,"name":"Juliana dos Santos Lupp","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"dos Santos","lastName":"Lupp","suffix":""},{"id":615231481,"identity":"bacad1bc-5745-48c5-ab5b-f0e352b28574","order_by":6,"name":"Talal Al-Nahlawi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYFAC5gaGBwwMMgwMPIwHGBgk5EBiBx7g0cDDwNjAkACigQikxRisJYEELQyJDSBhfFrs2Q82fkhgsOMxZz974MDPNov0+WGHHwJtsZPTbcBhC09is0QCQzKPZU9ewsHeNoncjbfTDIBako3NDuByWGIDUAszj8GBHIMDvNuAWmYngLQcSNyGSwv/w+YfCQz1PAbn3xgc/LtNIt1wdvoH/FokEtuAthzmMbiRY3AYaEuCvHQOAVtuPGyzSDA4DtTyxuCw7D8Jww3SOQUHEgxw+4W9P/nwjQ8V1XIG53MMH745UycvPzt984cPFXZyuLRAgAEy+wC6CEEg30CK6lEwCkbBKBgJAABLu2JxzycuFwAAAABJRU5ErkJggg==","orcid":"","institution":"Syrian Private University","correspondingAuthor":true,"prefix":"","firstName":"Talal","middleName":"","lastName":"Al-Nahlawi","suffix":""},{"id":615231482,"identity":"619fbc97-cce6-416c-a547-09b0949b14d9","order_by":7,"name":"Luciane Dias de Oliveira","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Luciane","middleName":"Dias","lastName":"de Oliveira","suffix":""},{"id":615231483,"identity":"b91599d5-a1e1-4ed0-a911-22fe8e191e91","order_by":8,"name":"Amjad Abu Hasna","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Amjad","middleName":"Abu","lastName":"Hasna","suffix":""}],"badges":[],"createdAt":"2026-03-21 15:54:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9186924/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9186924/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105954901,"identity":"21cd162a-e7a6-49e0-8f51-a5ec92502719","added_by":"auto","created_at":"2026-04-01 19:46:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":718197,"visible":true,"origin":"","legend":"\u003cp\u003eCellular viability of RAW 264.7 and MG63 cells after 5 min and 24h treatment with the experimental groups. Different uppercase letters indicate a statistically significant difference among the experimental groups.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9186924/v1/083d2ca731a268f0efa40032.jpg"},{"id":106093499,"identity":"033eb408-7ff2-4c94-ae7d-56612fac8e6f","added_by":"auto","created_at":"2026-04-03 11:37:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271859,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of micronuceli in RAW 267.7 and MG63 cells after 24h treatment with the experimental groups. Different uppercase letters indicate a statistically significant difference among the experimental groups.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9186924/v1/1233dee3840645104f82ce20.jpg"},{"id":105954902,"identity":"efe2b749-9463-43a3-af27-ee31ab048421","added_by":"auto","created_at":"2026-04-01 19:46:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":694768,"visible":true,"origin":"","legend":"\u003cp\u003eCytokines produced (in pg/mL) after conditioning RAW 264.7 with LPS and treatment with the experimental groups. Different uppercase letters indicate a statistically significant difference among the experimental groups.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9186924/v1/7d619fe18450344951ca2305.jpg"},{"id":105954904,"identity":"bad731af-b142-4570-bd93-f2281ddff24d","added_by":"auto","created_at":"2026-04-01 19:46:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":672494,"visible":true,"origin":"","legend":"\u003cp\u003eCytokines produced (in pg/mL) after conditioning MG63 with LPS and treatment with the experimental groups. Different uppercase letters indicate a statistically significant difference among the experimental groups.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9186924/v1/cafa4ac803eb5174d6723459.jpg"},{"id":106961026,"identity":"487e644d-64be-4392-b5d1-4ee158dbb68d","added_by":"auto","created_at":"2026-04-15 09:24:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3050221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9186924/v1/430c4612-7726-4f76-b507-b831bb572a9d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxicity, Genotoxicity and Cytokines Modulation of N-acetylcysteine, Calcium Hydroxide, Chlorhexidine and their Combinations","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEndodontic infections are characterized by a complex polymicrobial etiology in which diverse bacterial species and their associated pathogen-associated molecular patterns (PAMPs), including lipoteichoic acid (LTA) and lipopolysaccharide (LPS), play a critical role \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The presence of microorganisms and their metabolic by-products activates the host immune response, initiating an inflammatory cascade primarily mediated by the innate immune system. This early response involves neutrophils, macrophages, dendritic cells, and activation of the complement system, and is characterized by phagocytosis and the release of pro-inflammatory cytokines and chemokines. However, persistent microbial invasion subsequently promotes the activation of the adaptive immune response, characterized by the recruitment and activation of T and B lymphocytes to enhance antigen-specific immune regulation and contribute to the containment and modulation of the infection \u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA complex cytokine network is involved to regulate the inflammatory response including pro-inflammatory cytokines like interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) which play critical roles in promoting osteoclast differentiation and activation through upregulation of RANKL expression \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. IL-8 functions as a potent neutrophil chemoattractant, contributing to the maintenance of the acute inflammatory infiltrate, whereas IL-12 supports T-helper 1 (Th1) polarization and sustains a cell-mediated inflammatory response \u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Chemokines such as monocyte chemoattractant protein-1 (MCP-1) further regulate recruitment of monocytes and macrophages, reinforcing the chronic inflammatory environment \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Additionally, interferon-gamma (IFN-γ) contributes to macrophage polarization and perpetuation of the pro-inflammatory phenotype \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Conversely, regulatory cytokines such as interleukin-10 (IL-10) act to suppress excessive production of pro-inflammatory mediators, limit osteoclastogenic signaling, and contribute to immune balance and tissue repair \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The dynamic interplay between pro-inflammatory and regulatory mediators ultimately determines whether periapical lesions progress or shift toward resolution, influenced by bacterial virulence, host immune regulation and the efficacy of the treatment.\u003c/p\u003e \u003cp\u003eRoot canal treatment, in turn, is indicated to combat the invasion of the bacteria and their by-products, and to control the inflammatory process using files, endodontic irrigants and eventually intracanal medications like calcium hydroxide [Ca(OH)₂], which is commonly used because of its antimicrobial action, ability to neutralize endotoxins, ability to induce tissue repair through the deposition of mineralized tissue, and having a good tolerance by periapical tissues \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Ca(OH)₂ can reduce the production of pro-inflammatory cytokines, such as IL-1β and IL-6, and has the capacity to neutralize the cytotoxic effects of LPS and LTA \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Besides to, chlorhexidine digluconate (CHX) which is also widely used intracanal medication because of its biocompatibility and broad spectrum of antimicrobial activity \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, besides to its efficacy in reducing different pro-inflammatory cytokines \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eN-acetylcysteine (NAC) is a derivative of L-cysteine, with recognized mucolytic and antioxidant action, acting as a precursor of glutathione and contributing to the neutralization of free radicals. Although it is not an antibiotic medication, it presents antimicrobial properties and the ability to reduce biofilm formation by inhibiting the production of extracellular polysaccharides, in addition to promoting the disruption of mature biofilms and decreasing bacterial adhesion \u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. NAC also presents an inhibitory effect on the expression of several pro-inflammatory cytokines \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e increases the levels of Resolvin \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, and has been indicated as a promising intracanal medication, as it demonstrates activity against relevant endodontic pathogens \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, NAC, Ca(OH)₂, CHX, and their combinations have not yet been evaluated regarding their cytotoxicity, genotoxicity and effects on cytokine production. Therefore, the aim of this study was to assess the cytotoxicity, genotoxicity, and anti-inflammatory potential of these intracanal medications, alone, and in combination. The null hypothesis tested was that these medications would not be biocompatible and would not exert any anti-inflammatory effect.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample size calculation\u003c/h2\u003e \u003cp\u003eIt was performed using the online calculator: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://estatistica.bauru.usp.br/calculoamostral/calculos.php\u003c/span\u003e\u003cspan address=\"http://estatistica.bauru.usp.br/calculoamostral/calculos.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e with the following parameters: 8 groups, an estimated standard deviation of 2, a minimum detectable difference of 3.6, α\u0026thinsp;=\u0026thinsp;5%, and β\u0026thinsp;=\u0026thinsp;20% (80% power). The minimum required sample size was determined to be 10 per group (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental groups\u003c/h3\u003e\n\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eControl group: saline solution (Eurofarma, S\u0026atilde;o Paulo, SP, Brazil);\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCa(OH)₂: The Ca(OH)₂ powder (Biodin\u0026acirc;mica Qu\u0026iacute;mica e Farmac\u0026ecirc;utica LTDA, Paran\u0026aacute;, Brazil) was with sterile saline solution (1:1 ratio, 100 \u0026micro;g of powder and 100 \u0026micro;L of sterile saline solution).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCHX: 100 \u0026micro;L of 2% chlorhexidine gel (Terap\u0026ecirc;utica Farm\u0026aacute;cia de Manipula\u0026ccedil;\u0026atilde;o, S\u0026atilde;o Jos\u0026eacute; dos Campos, SP, Brazil);\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNAC: The NAC powder (Merck KGaA, Darmstadt, Germany) was mixed with sterile saline solution (1:1 ratio, 100 \u0026micro;g of powder and 100 \u0026micro;L of sterile saline solution).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNAC\u0026thinsp;+\u0026thinsp;Ca(OH)₂: The NAC and Ca(OH)₂ powders was mixed with sterile saline solution (0.5:0.5:1 ratio, 50 \u0026micro;g of NAC powder\u0026thinsp;+\u0026thinsp;50 \u0026micro;g of Ca(OH)₂ powder and 100 \u0026micro;L of sterile saline solution).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNAC\u0026thinsp;+\u0026thinsp;CHX: The NAC powder was mixed with CHX gel (1:1 ratio, 100 \u0026micro;g of powder and 100 \u0026micro;L of CHX gel)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCa(OH)₂ + CHX: The Ca(OH)₂ powder was mixed with CHX gel (1:1 ratio, 100 \u0026micro;g of powder and 100 \u0026micro;L of CHX gel).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNAC\u0026thinsp;+\u0026thinsp;Ca(OH)₂ + CHX: The NAC and Ca(OH)₂ powders was mixed with CHX gel (0.5:0.5:1 ratio, 50 \u0026micro;g of NAC powder\u0026thinsp;+\u0026thinsp;50 \u0026micro;g of Ca(OH)₂ powder and 100 \u0026micro;L of CHX gel).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn a 24-well plate, the medications were prepared according to the previously established proportion. Each well was filled with 2 mL of Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Invitrogen, New York, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% amphotericin B, and kept in an incubator with 5% CO₂ at 37\u0026deg;C for 24 h. Later, 100 \u0026micro;L of the conditioned medium previously in contact with each medication was used as a treatment and transferred to the corresponding wells containing cultured cells according to the respective experimental group.\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eMurine macrophage cells (RAW 264.7) and human osteoblasts-like cells (MG-63) were used, obtained from the Cell Bank of Rio de Janeiro (APABCAM, RJ, Brazil). The cells were cultured in supplemented DMEM and maintained in an incubator at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. The culture medium was replaced every 48 h until the required number of cells was obtained.\u003c/p\u003e \u003cp\u003eFor the experiments, RAW 264.7 cells were mechanically detached from the bottom of the flasks using a cell scraper, whereas MG-63 cells were chemically detached with 0.25% trypsin-EDTA solution (Cultilab, Campinas, SP, Brazil). The quantification of viable cells was performed using the Trypan blue exclusion test (0.4%, Sigma-Aldrich, St. Louis, MO, USA).\u003c/p\u003e\n\u003ch3\u003eCytotoxicity evaluation – MTT colorimetric assay\u003c/h3\u003e\n\u003cp\u003eThe viable cells (2 \u0026times; 10⁴) were cultivated in 96-well microplates at 37\u0026deg;C with 5% CO₂ for 24 h to allow cell adhesion. After incubation, the culture medium was removed, and the wells were gently washed with sterile phosphate-buffered saline (PBS) (Cultilab, Campinas, SP, Brazil). The cells were treated with 100 \u0026micro;L of the conditioned medium of the respective intracanal medication for 5 mins or 24 h being incubated under the same conditions (37\u0026deg;C, 5% CO₂).\u003c/p\u003e \u003cp\u003eLater, cell viability was determined by assessing mitochondrial metabolic activity through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystals by viable cells. The MTT solution was prepared by dissolving 0.5 mg of MTT powder (Sigma-Aldrich, Germany) in 1 mL of sterile PBS. Subsequently, 100 \u0026micro;L of MTT solution was added to each well, and the plates were incubated for 4 h at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂, protected from light. After incubation, the MTT solution was carefully discarded, and 100 \u0026micro;L of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well to solubilize the formazan crystals formed by viable cells. The plates were incubated for 10 min and gently shaken for an additional 10 min to ensure complete dissolution.\u003c/p\u003e \u003cp\u003eThe absorbance was measured at 570 nm using a microplate reader. Optical density (OD) values were converted into percentage of cell viability using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Cell\\:viability\\:=\\frac{\\:OD\\:of\\:treated\\:group\\:\\times\\:\\:100}{Mean\\:of\\:OD\\:of\\:control\\:group}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eGenotoxicity evaluation - cytokinesis-block micronucleus assay\u003c/h3\u003e\n\u003cp\u003eDifferently, the viable cells were seeded in 24-well plates at a density of 5 \u0026times; 10⁵ cells/mL and incubated for 24 h at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. The cells were treated with 100 \u0026micro;L of the conditioned medium of the respective intracanal medication for 24 h being incubated under the same conditions (37\u0026deg;C, 5% CO₂). Ethyl methane sulfonate (EMS) was used as a control group because of its capacity in forming micronuclei.\u003c/p\u003e \u003cp\u003eSubsequently, cytochalasin B (Sigma-Aldrich) was added at a final concentration of 6 \u0026micro;g/mL, and the cells were incubated for an additional 24 h at 37\u0026deg;C with 5% CO₂ to inhibit cytokinesis and allow the formation of binucleated cells. Then, the cells were subjected to hypotonic treatment and fixed with a methanol:acetic acid solution (3:1) for 10 minutes. The fixation procedure was repeated three times. The wells were then stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (Sigma, Missouri, USA), and the dye was removed after 5 min of contact with the cells and then washed with PBS.\u003c/p\u003e \u003cp\u003eMicronucleus analysis was performed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) at 400\u0026times; magnification. A total of 2,000 cells per well were evaluated in at least two independent experiments. Micronuclei were identified as small DNA-containing structures located in the cytoplasm, clearly separated from the main nucleus, surrounded by a nuclear membrane, and with an area smaller than one-third of the main nucleus. Only cells containing fewer than five micronuclei were included in the analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnti-inflammatory evaluation - Cytometric Bead Array\u003c/h2\u003e \u003cp\u003eThe viable cells were seeded into 24-well plates at a density of 1 \u0026times; 10⁵ cells per well and incubated for 24 h to allow cell adhesion. The cells were treated with 100 \u0026micro;L of the conditioned medium of the respective intracanal medication for 24 h. Simultaneously, LPS of \u003cem\u003eEscherichia coli\u003c/em\u003e (Sigma-Aldrich, St. Louis, MO, USA) was added to each well at a final concentration of 5 \u0026micro;g/mL to induce an inflammatory response. The plates were then incubated for 24 h at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e \u003cp\u003eLater, the supernatants were collected and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until cytokine quantification. For RAW 264.7 cells, IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70 were quantified using the Mouse Inflammation Kit (BD Biosciences). For MG-63 cells, cytokine levels (IL-8, IL-1β, IL-6, IL-10, TNF, and IL-12p70) were measured using the Human Inflammatory Cytokine Kit (BD Biosciences, San Diego, CA, USA). Cytokine analysis for both cell lines was performed by flow cytometry using the Cytometric Bead Array (CBA) system, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were tabulated and submitted to Shapiro\u0026ndash;Wilk normality test. Then, non-parametric data were analyzed by Kruskal Wallis and complemented by Dunn for multiple comparisons. Whereas parametric data were analyzed by \u003cem\u003eone-way Anova\u003c/em\u003e and complemented by \u003cem\u003eTukey\u003c/em\u003e for multiple comparisons. All these tests were performed considering a significant level of α\u0026thinsp;\u0026le;\u0026thinsp;0.05 using GraphPad Prism 10.6.1 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity evaluation\u003c/h2\u003e \u003cp\u003eAfter 5 min of treatment, all the experimental groups presented cellular viability of RAW 264.7 cells without a statistically significant difference with the control group except of Ca(OH)\u003csub\u003e2\u003c/sub\u003e, CHX, NAC\u0026thinsp;+\u0026thinsp;Ca(OH)\u003csub\u003e2\u003c/sub\u003e, and NAC\u0026thinsp;+\u0026thinsp;Ca(OH)\u003csub\u003e2\u003c/sub\u003e + CHX groups that promoted the cellular viability more than saline solution control group with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) up to 92.4, 263.53, 92.67 and 252.38%, respectively. Nevertheless, after 24 h of treatment, all the experimental groups presented cellular viability without a statistically significant difference with the control group except of NAC, NAC\u0026thinsp;+\u0026thinsp;CHX and Ca(OH)\u003csub\u003e2\u003c/sub\u003e + CHX groups that presented lower values of viable cells (-19.1, -14.42 and \u0026minus;\u0026thinsp;19.12, respectively) with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, after 5 min and 24 h of treatment, all the experimental groups presented cellular viability of MG63 cells without a statistically significant difference with the control group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) except of NAC group after 5 min that reduced the cellular viability to \u0026minus;\u0026thinsp;14.32% and Ca (OH)\u003csub\u003e2\u003c/sub\u003e + CHX after 24 h that promoted the cellular viability with 35.20% with a statistically significant difference with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenotoxicity evaluation\u003c/h2\u003e \u003cp\u003eAfter 24 h of treatment, all the experimental groups presented a relatively lower number of micronuclei in RAW 264.7 and MG63 cells in comparison with the EMS group with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Except of CHX, which presented a high number of micronuclei in RAW 264.7 cells being as genotoxic as EMS statistically as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnti-inflammatory evaluation\u003c/h2\u003e \u003cp\u003eIn RAW 264.7, NAC group induced the production of high values of IL-12p70 with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with all the experimental groups, of IL-10 with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with all the experimental groups except of NAC\u0026thinsp;+\u0026thinsp;CA(OH)\u003csub\u003e2\u003c/sub\u003e and NAC\u0026thinsp;+\u0026thinsp;CHX groups, and of IFN-γ with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with all the experimental groups except of NAC\u0026thinsp;+\u0026thinsp;CHX group. Differently, Ca(OH)\u003csub\u003e2\u003c/sub\u003e group induced the production of high values of TNF with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with all the experimental groups except of NAC and Ca(OH)\u003csub\u003e2\u003c/sub\u003e + CHX group, and of MCP-1 with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with all the experimental groups. Nevertheless, NAC, Ca(OH)\u003csub\u003e2\u003c/sub\u003e and NAC\u0026thinsp;+\u0026thinsp;CHX groups induced the production of high values IL-6 with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn MG63, NAC induced the production of high values IL-12p70, TNF, IL-10 and IL-1β, with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with all the experimental groups. In addition, NAC and Ca(OH)\u003csub\u003e2\u003c/sub\u003e + CHX group induced the production of high values IL-6 with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in comparison with all the experimental groups except of CHX which was statistically similar. Differently, Ca(OH)\u003csub\u003e2\u003c/sub\u003e, CHX, Ca(OH)\u003csub\u003e2\u003c/sub\u003e + CHX groups induced the production of high values IL-8 with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in comparison with all the experimental groups as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study evaluated the cytotoxicity, genotoxicity, and inflammatory response\u0026ndash;modulating potential of NAC, Ca(OH)₂, CHX and their combinations on RAW 264.7 macrophages and MG-63 osteoblast-like cells. Overall, the results showed that some medications demonstrated acceptable biocompatibility, whereas others exhibited potential genotoxic effects or modulated the production of pro- and anti-inflammatory cytokines. Therefore, the null hypothesis was partially rejected.\u003c/p\u003e \u003cp\u003eIn the present study, all tested intracanal medications demonstrated high cellular viability of MG63 after treatment for 5 min and 24 h being as biocompatible as saline solution except of NAC after 5 min that reduced the cellular viability by 14.32%. Although NAC slightly reduced the cellular viability of MG63 at the 5-minute time point, cell viability after 24 h was comparable to that observed with the other tested medicaments. In the literature, NAC was added to zinc oxide\u0026ndash;eugenol-based materials to improve their biocompatibility in MG63 \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Otherwise, no significant reductions in cell viability were observed. This align with the results in the literature that confirm the biocompatibility of Ca(OH)\u003csub\u003e2\u003c/sub\u003e, CHX and their combination in osteoblasts \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e T This suggests that, under the experimental conditions of the present study, the tested materials did not directly compromise the metabolic activity of cells involved in bone repair processes. From a biological perspective, this observation is particularly relevant, as osteoblasts play a central role in bone formation, resolution of periapical lesions, and modulation of the local inflammatory microenvironment \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e Therefore, the maintenance of MG-63 cell viability indicates that the tested medications did not significantly interfere with the basal metabolic activity of these cells, thereby preserving their potential to contribute to tissue repair.\u003c/p\u003e \u003cp\u003eIn contrast, RAW 264.7 cells exhibited a response that was dependent on exposure time. After 5 minutes, all experimental groups showed high biocompatibility, in some cases exceeding that observed in the saline solution, particularly in the Ca(OH)₂, CHX, NAC\u0026thinsp;+\u0026thinsp;Ca(OH)₂, and NAC\u0026thinsp;+\u0026thinsp;Ca(OH)₂ + CHX groups. As highly adaptable cells, macrophages are capable of rapidly adjusting their energy metabolism in response to chemical and inflammatory stimuli \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Considering that the MTT assay evaluates the activity of the mitochondrial enzyme succinate dehydrogenase, alterations in cellular metabolic state may directly influence the observed viability indices \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe reduction in viability observed after 24 h in the NAC, NAC\u0026thinsp;+\u0026thinsp;CHX, and Ca(OH)₂ + CHX groups may be associated with redox imbalance or prolonged metabolic stress. CHX has been reported to induce mitochondrial dysfunction and alterations in cell membrane integrity in a manner dependent on concentration and exposure time \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Although NAC is widely recognized for its antioxidant properties, it may also modulate signaling pathways involving reactive oxygen species (ROS) at certain concentrations, potentially affecting cellular dynamics \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Additionally, the Ca(OH)₂ + CHX association deserves attention. While Ca(OH)₂ is generally considered biocompatible and capable of inducing mineralization, its high pH may cause transient alterations in cellular homeostasis and may also affect the chemical stability of CHX in highly alkaline environments \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding genotoxicity, the results revealed distinct behaviors among the tested medications. A significant increase in micronucleus number was observed in the groups containing CHX, particularly in RAW 264.7, whereas NAC and Ca(OH)₂ did not promote relevant genomic damage under the experimental conditions, however, all tested medications presented a relatively low number of micronuclei in MG63. The presence of micronuclei indicates chromosomal breaks or errors in chromosome segregation during mitosis; events often associated with oxidative stress. Under such conditions, ROS can induce oxidative lesions that, if not properly repaired, may compromise cellular genomic stability \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that CHX can induce oxidative stress and DNA damage in a dose-dependent manner, increasing ROS production, and activating apoptotic pathways \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. These mechanisms are consistent with the increased micronucleus frequency observed in the present study, particularly in inflammatory cells, which typically exhibit higher basal ROS production. In contrast, although Ca(OH)₂ presents a highly alkaline pH, it did not significantly increase micronucleus formation under the experimental conditions, suggesting that its alkalinity alone was not genotoxic in this model, this was affirmed over stem cells of apical papilla in a previous study \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Furthermore, NAC demonstrated a stable biological profile as it can reduce oxidative DNA damage and improving cellular resistance to genotoxic stress by scavenging ROS and supporting intracellular glutathione levels \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, RAW 264.7 and MG63 cells were stimulated with \u003cem\u003eE. coli\u003c/em\u003e LPS to mimic an inflammatory microenvironment. Therefore, cytokine production should be interpreted in the context of LPS-induced cells activation and the modulatory effects of the tested medications \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Under these conditions, NAC increased the production of the pro-inflammatory cytokines IL-12p70, IL-6, and IFN-γ in RAW 264.7 cells. This response may be associated with the reduction in RAW 264.7 viability observed after 24 h in the NAC group, as decreased viability may reflect a state of metabolic or redox stress \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In such conditions, macrophages can activate inflammatory signaling pathways that contribute to increased cytokine production. Interestingly, NAC also increased the production of IL-10, an anti-inflammatory cytokine, suggesting that NAC may exert an immunomodulatory effect by balancing inflammatory activation and regulatory responses. In vitro study confirmed that NAC can adjust the production of mediators such as TNF-α, IL-1β, IL-6, and IL-8 in activated macrophages, refining the final inflammatory response \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDifferently, Ca(OH)₂ induced the production of only pro-inflammatory cytokines, including TNF-α, IL-6, and MCP-1. These findings are consistent with a previous study reporting that Ca(OH)₂ stimulates the production of TNF-α in macrophages exposed to LTA from \u003cem\u003eEnterococcus faecalis\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the same study demonstrated that the combination of Ca(OH)₂ and CHX also promoted TNF-α production, which agrees with the results observed in the present study. This effect may be related to the high alkalinity of Ca(OH)₂, which can activate macrophages and influence inflammatory signaling pathways.\u003c/p\u003e \u003cp\u003eAnother finding that deserves attention is the cytokine profile observed in the NAC\u0026thinsp;+\u0026thinsp;CHX group, which showed increased production of IL-10, IL-6, and IFN-γ. This pattern suggests that the addition of NAC to CHX may exert a modulatory effect on the inflammatory response, potentially contributing to a more balanced regulation between pro- and anti-inflammatory signaling.\u003c/p\u003e \u003cp\u003eIn MG-63 cells, a consistent increase in the production of IL-12p70, TNF-α, IL-10, IL-6, and IL-1β was observed following treatment with NAC compared with the other experimental groups. This finding suggests that NAC directly influenced the functional activity of these osteoblast-like cells while preserving their potential for tissue repair \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. The concomitant increase in IL-10 indicates that the response induced by NAC was not exclusively pro-inflammatory but also involved regulatory mechanisms controlling the inflammatory response. IL-10 is recognized as an important anti-inflammatory mediator, as it can limit the production of pro-inflammatory cytokines and prevent excessive tissue damage \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Considering the ability of NAC to modulate intracellular redox status, the observed changes in cytokine production may be associated with alterations in ROS-dependent signaling pathways. Such modulation may influence cellular processes involved in periapical bone repair, highlighting the potential relevance of NAC in regulating the inflammatory microenvironment associated with periapical tissues.\u003c/p\u003e \u003cp\u003eThe combinations of the tested medications NAC\u0026thinsp;+\u0026thinsp;Ca(OH)\u003csub\u003e2\u003c/sub\u003e, NAC\u0026thinsp;+\u0026thinsp;CHX, Ca(OH)\u003csub\u003e2\u003c/sub\u003e +CHX and NAC\u0026thinsp;+\u0026thinsp;Ca(OH)\u003csub\u003e2\u003c/sub\u003e +CHX generally did not produce marked alterations in the levels of pro- or anti-inflammatory cytokines, except for a few isolated findings. Overall, these combinations also demonstrated acceptable biocompatibility and did not significantly increase micronucleus formation.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe tested medications maintained high cellular viability of RAW 264.7 cells after 5 min of exposure and MG-63 cells after 24 h.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe medications exhibited low genotoxic potential, with a low frequency of micronuclei formation in most groups, except for CHX in RAW 264.7 cells.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNAC demonstrated a potential immunomodulatory effect by promoting a balance between pro-inflammatory activation and regulatory cytokine responses.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors deny any conflicts of interest related to this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was financially supported by S\u0026atilde;o Paulo Research Foundation (FAPESP) 2023/10158-2 and by the Institutional Scholarship Program for Scientific Initiation (PIBIC) of the National Council for Scientific and Technological Development (CNPq).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., T.A-N., L.D.O., and A.A.H.; Data curation: A.C.F.S., S.M.S.S., L.D.O., and A.A.H.; Formal analysis: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Funding acquisition: V.A.M., and A.A.H.; Investigation: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Methodology: A.C.F.S., S.M.S.S., T.A-N., L.D.O., and A.A.H.; Project administration: A.A.H.; Resources: L.D.O., and A.A.H.; Software: L.D.O., and A.A.H.; Supervision: T.A-N., L.D.O., and A.A.H.; Validation: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Visualization: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., J.S.L., L.D.O., and A.A.H.; Roles/Writing - original draft: A.C.F.S, S.M.S.S., D.R., V.A.M., L.S.C., and J.S.L.; and Writing - review \u0026amp; editing: T.A-N., L.D.O., and A.A.H.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available in the UNESP repository, [https://hdl.handle.net/11449/318572](https:/hdl.handle.net/11449/318572)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede \u0026emsp;\u0026ensp;\u0026ensp;Gomes, B. P. F., Herrera, D. R. \u0026amp; A. \u0026amp; Etiologic role of root canal infection in apical periodontitis and its relationship with clinical symptomatology. \u003cem\u003eBraz Oral Res.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, e69 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorse, D. R. Immunologic aspects of pulpal-periapical diseases. \u003cem\u003eOral Surg. Oral Med. Oral Pathol.\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e, 436\u0026ndash;451 (1977).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBergenholtz, G. Pathogenic mechanisms in pulpal disease. \u003cem\u003eJ. Endod\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 98\u0026ndash;101 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiqueira, J. F. \u0026amp; R\u0026ocirc;\u0026ccedil;as, I. N. Bacterial pathogenesis and mediators in apical periodontitis. \u003cem\u003eBraz Dent. 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The structural network of Interleukin-10 and its implications in inflammation and cancer. \u003cem\u003eBMC Genom.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (Suppl 4), S2 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"acetylcysteine, calcium hydroxide, chlorhexidine, Cytotoxicity, Genotoxicity, Cytokines","lastPublishedDoi":"10.21203/rs.3.rs-9186924/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9186924/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to evaluate the cytotoxicity, genotoxicity and effect on cytokine production of n-acetylcysteine (NAC), calcium hydroxide [Ca(OH)₂], chlorhexidine (CHX) and their combinations on murine macrophage cells (RAW 264.7) and human osteoblasts-like cells (MG-63). Cytotoxicity was evaluated by MTT colorimetric assay after 5 min and 24 h. Genotoxicity evaluation was evaluated by cytokinesis-block micronucleus assay. In addition, cytometry bead arrays to quantify different cytokine production by RAW 264.7 and MG63 cells. Data were statistically analyzed with a significant level of α\u0026thinsp;\u0026le;\u0026thinsp;0.05. All medications promoted high cellular viability of RAW 264.7 after 5 min, and of MG63 after 24h. All medications presented a low number of micronuclei except for CHX in Raw 264.7. NAC was highlighted as it stimulates high production of IL-12p70, IL-6, and IFN-γ in RAW 264.7 cells, in addition to IL-12p70, TNF-α, IL-10, IL-6, and IL-1β in MG63. Therefore, NAC demonstrated a potential immunomodulatory effect by promoting a balance between pro-inflammatory activation and regulatory cytokine responses.\u003c/p\u003e","manuscriptTitle":"Cytotoxicity, Genotoxicity and Cytokines Modulation of N-acetylcysteine, Calcium Hydroxide, Chlorhexidine and their Combinations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 19:46:24","doi":"10.21203/rs.3.rs-9186924/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43fae2dd-b3f3-4987-aae0-7923b0572c88","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65463982,"name":"Biological sciences/Biochemistry"},{"id":65463983,"name":"Biological sciences/Cell biology"},{"id":65463984,"name":"Biological sciences/Drug discovery"},{"id":65463985,"name":"Biological sciences/Immunology"},{"id":65463986,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-14T06:42:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 19:46:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9186924","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9186924","identity":"rs-9186924","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}