Biological effects of N-acetylcysteine, calcium hydroxide, chlorhexidine and their combinations as intracanal medications: cytotoxicity, genotoxicity and cytokine modulation.

OBJECTIVE

This study aimed to evaluate whether the combination of N-acetylcysteine (NAC), a compound with antimicrobial, antioxidant, and immunomodulatory properties, with conventional intracanal medications calcium hydroxide [Ca(OH)₂] and chlorhexidine (CHX) alters their biological effects on murine macrophage cells (RAW 264.7) and human osteoblasts-like cells (MG-63), with particular emphasis on cytotoxicity, genotoxicity, and cytokine production.

MATERIALS AND

METHODS

The study groups were control group of Dulbecco's Modified Eagle Medium (DMEM), Ca(OH)₂ (100 mg/100 µL saline solution), 2% CHX gel (100 µL), NAC (100 mg/100 µL saline solution), NAC + Ca(OH)₂ (50 + 50 mg/100 µL saline solution), NAC + CHX (100 mg/100 µL 2% CHX gel), Ca(OH)₂ + CHX (100 mg/100 µL 2% CHX gel), and NAC + Ca(OH)₂ + CHX (50 + 50 mg/100 µL 2% CHX gel). The preparations were conditioned in culture medium for 24 h, and 100 µL of the resulting conditioned medium was used for cell exposure. Cytotoxicity was evaluated by MTT colorimetric assay that measures the metabolic activity of the cells after 5 min and 24 h. Genotoxicity was evaluated by cytokinesis-block micronucleus assay after 24 h with Ethyl methanesulfonate (EMS) as control group. In addition, cytometry bead arrays to quantify different cytokine production by RAW 264.7 and MG63 cells under LPS-stimulated conditions. Data were statistically analyzed with a significant level of α ≤ 0.05.

RESULTS

Most experimental groups maintained metabolic activity comparable to the control group in both cell lines, although NAC, NAC + CHX, and Ca(OH)₂ + CHX reduced RAW 264.7 metabolic activity after 24 h. Most experimental groups showed significantly lower micronucleus frequency in RAW 264.7 and MG-63 cells compared with the EMS positive control group (P < 0.0001). However, the CHX group in RAW 264.7 cells exhibited a micronucleus frequency that was not significantly different from the EMS group. Still, NAC increased IL-12p70, IL-6, IFN-γ, and IL-10 production in RAW 264.7, and increased IL-12p70, TNF-α, IL-10, IL-6, and IL-1β levels in MG-63.

CONCLUSION

NAC modulated cytokine production in both cell models under LPS-stimulated conditions, resulting in a mixed profile of pro-inflammatory and regulatory mediators rather than a purely anti-inflammatory response. Most tested medications maintained acceptable metabolic activity and low genotoxic potential under the evaluated conditions, although CHX increased micronucleus formation in RAW 264.7 cells.