The effect of reduced glutathione on the toxicity of silver diamine fluoride in rat pulpal cells

Abstract Introduction Due to its ability to arrest untreated dental caries, silver diamine fluoride (SDF) has been advocated for indirect pulp capping procedures. However, the high concentrations of silver and fluoride in SDF raise concerns about its biocompatibility to pulpal tissues. Objectives This study aimed to investigate the effect of SDF on the viability, alkaline phosphatase (ALP) activity, and morphology of pulpal-like cells (RPC-C2A) and to evaluate the influence of reduced glutathione (GSH) on SDF-induced cytotoxicity and deposit formation on dentin. Methodology The cytotoxicity of diluted 38% SDF solutions (10-4 and 10-5), with or without the addition of 5 mM or 50 mM GSH, was evaluated at 6 and 24 hours. Cell viability was detected using WST-8 and the effect on ALP activity was performed using an ALP assay kit. Cell morphology was observed using a phase-contrast microscope. Scanning electron microscopy analysis was conducted to evaluate the effect of GSH incorporation or conditioning on SDF-induced deposit formation on dentin discs. Cytotoxicity data were analyzed by two-way analysis of variance (ANOVA) and Tukey post hoc tests (p<0.05). Results There were significant differences between the groups. The results demonstrated that all tested SDF dilutions caused a remarkable cytotoxic effect, while the addition of GSH prevented SDF-induced damage at 6-hour exposure time in the higher dilution of SDF. Dentin treated with plain SDF or GSH-incorporated SDF solution showed deposit formation with occluded dentinal tubules, unlike the other groups. Conclusion SDF severely disturbed the viability, mineralization-ability, and morphology of pulpal-like cells, while controlled concentrations of GSH had a short-term protective effect against SDF-induced damage. GSH showed an inhibitory effect on SDF-induced dentinal deposit formation. Further research is warranted to evaluate the effect of GSH on caries-arresting, anti-hypersensitivity, and antibacterial functions of SDF.


Introduction
Silver diamine fluoride (SDF), an alkaline colorless solution composed of 24-29% silver (Ag) and 5-6% fluoride (F), is used for arresting caries progression and treating dentin hypersensitivity. This agent is gaining considerable attention due to the promising results of both clinical and laboratory studies. 1 The mechanisms of action are still not clearly understood.
However, it is believed that Ag ions provide antibacterial properties against some cariogenic bacteria, and F ions enhance remineralization. 2,3 SDF also forms insoluble precipitates with calcium and phosphate that physically block dentinal tubules, minimizing dentin hypersensitivity. 4 SDF application is considered a simple, inexpensive, and non-invasive procedure.
Nevertheless, it can lead to discoloration of teeth. 5 The Ag ions can infiltrate within demineralized and the underlying mineralized dentin. 6 Ag and F ions can penetrate up to 0.2 mm into dentin. 7 Despite the popularity of SDF, some reports suggest a cautious application of this solution into the oral cavity. 8,9 Compared to silver chloride, SDF has higher toxicity against osteoblast-like cells and human gingival fibroblasts (HGF) -only one hour of contact with HGF induced irreversible cell death by necrosis. 8 F concentration (44,800 ppm) in SDF is considered one of the highest compared to other available products. 10 The high concentrations of Ag and F raise concerns about their harmful effects on pulpal tissue. The cytotoxic effect of SDF on HGF occurred even at low concentrations (0.1%) and lasted for nine weeks even after rinsing SDF-treated dentin discs with saline. 9 Despite inadequate investigations on the cytotoxic effect of SDF on pulpal cells, studies show the potential use of SDF in indirect pulp capping procedure in deep cavities and gained much attention due to SDF's ability to induce tertiary dentin formation. 11 Glutathione is a low molecular weight thiolcompound that contains a sulfhydryl group in its structure. It has an essential role as an antioxidant that functions by various mechanisms such as metal chelators and radical quenchers. 12,13 Glutathione is possibly the most prevalent and most important intracellular thiol-disulfide redox buffer in mammalian cells. The active form of glutathione is called reduced glutathione (GSH), a water-soluble tripeptide containing cysteine, glutamic acid, and glycine that contributes to the intracellular non-protein thiols. [14][15][16] Ag cytotoxicity on rat and human liver cells could partially result from GSH depletion. 17,18 The strong affinity of Ag to the sulfhydryl group could be responsible for the observed GSH depletion. 19 Moreover, GSH is involved in the detoxification of Ag from blood plasma. 20 F is also known to decrease the level of GSH, which reflects increased utilization of the latter due to the oxidative stress generated by the administration of F at a level of 25 ppm in the drinking water of rats. 21 The increased oxidative stress and the adverse effect of F on the antioxidant function have also been confirmed in several other studies. [22][23][24][25][26] There is a paucity of scholarly literature on the adverse effect of SDF on pulpal cells and preventing or minimizing this damage. Thus, this study aims to assess the effect of SDF on rat-pulpal cells, test the effect of GSH on SDF-induced damage on the cells as mentioned earlier, and its effect on the ability of SDF to form deposits on the surface of dentin discs. The Using a one-way ANOVA test, the sample size was calculated based on a significance level of 5% and power of 80% to detect a difference of 0.2 in spectrophotometric absorbance. The required sample size for each group is at least 4 (powerandsamplesize. The resultant experimental solutions were as the following (n=6): (I) SDF X 10 -4 (pH 8.2), (II) SDF X 10 -5 (pH 8.11), (III) SDF X 10 -4 + 5 mM GSH (pH 7.6), (IV) SDF X 10 -4 + 50 mM GSH (pH 6.12), (V) SDF X 10 -5 + 5 mM GSH (pH 7.58), and (VI) SDF X 10 -5 + 50 mM GSH (pH 6.3). Because of the strong alkaline property of SDF, we diluted SDF to a concentration equal or lower than SDF X 10 -4 to have a pH less than 8.0, which caused no precipitation or turbidity in the cell culture medium. Cell culture in fresh medium (pH 7.74) without experimental solution served as the control. After the incubation time, the culture medium was discarded, and cells were washed with 200 µL phosphate buffer solution to prevent any interaction between the test solutions and the colorimetric assay.
One hundred microliters of new culture medium were added to each well, and cell viability was measured

ALP activity measurement
Cultured RPC-C2A cells were treated with the same solutions described above for 6 or 24 h (n=6). ALP activity was determined by using ALP Assay Kit (Takara Bio, Shiga, Japan).
Scanning electron microscopy analysis of dentin discs treated with SDF According to the protocol approved by the Human Research Ethics Committee, extracted human noncarious third molars were used in this part of the study. Flat dentin discs were created perpendicular to the tooth's longitudinal axis, using a slow-speed diamond saw (Isomet Low Speed Saw; Buehler, Lake Bluff, IL, USA) under water lubrication. Dentin surfaces were polished using up to 4000-grit silicon-carbide paper under water irrigation, followed by sonication with deionized water for debris removal. The dentin discs were randomly divided into four groups. In group 1 (control group), dentin discs were treated with distilled water. In groups 2 and 3, 38% SDF (pH 10) was applied, followed by either distilled water or 20% GSH solution (pH 4), respectively. Group 4 discs were treated with a solution that contained 38% SDF and 20% GSH (pH 6.8-7.8). All solutions were applied with agitation using micro-brushes for 1 min.

Results
Effect on viability, ALP activity, and morphology of RPC-C2A cells    SEM analysis of dentin discs treated with SDF Dentin discs treated with 38% SDF followed by a rinse with distilled water showed marked mineral deposit formation on the surface along with occluded dentinal tubules (Figure 4b). Nevertheless, these findings were less prominent on dentinal surfaces treated with the solution that contained 38% SDF and 20% GSH, followed by a rinse with distilled water ( Figure 4c). Meanwhile, dentin discs treated with 38% SDF followed by conditioning with 20% GSH showed less deposit formation than the aforementioned experimental groups (Figure 4d). The control group showed the least amount of deposit formation ( Figure   4a).

Discussion
This study is the first to assess the effect of SDF on pulpal-like cells and the efficacy of GSH to mitigate SDF-induced toxicity. We rejected the null hypotheses because SDF adversely affected the viability, ALP activity and morphology of the used cells, and GSH at specific concentrations and exposure time mitigated One of the recently suggested SDF applications is its use in deep cavities for indirect pulp capping procedures due to the claimed ability of SDF to induce tertiary dentin formation. 11 Despite the established safety of SDF application in this procedure, there is always a concern about the effect of the released F and Ag ions 8,9, which can penetrate easily into dentin, especially within demineralized dentin left at the deepest part of the cavity preparation in indirect pulp procedures, 6,7 thus the need for a non-toxic indirect pulp capping agent cannot be stressed enough.
Due to the chances of pulpal cells exposure to SDF, pulpal-like cells were used in this study to assess its effect on the viability, ALP activity, and morphology of these cells. In a preliminary study, we used higher SDF concentrations in the cell culture medium than the concentrations used in this study. We noticed white turbidity and increased pH (>8) of cell culture medium at high SDF concentrations. Thus, in this study, 38% SDF was diluted at 10,000-fold (0.0038%) and 100,000-fold (0.00038%), which showed no turbidity in the culture medium. SDF at 0.0038% and 0.00038% adversely affected cell viability and changed cellular morphology from polygonal to round with loss of intercellular spaces. SDF also suppressed the ALP activity of the cells. ALP activity correlates with the mineralization ability of the cells. 29 Thus, it is essential to maintain this activity for successful indirect pulp therapy procedure by the formation of tertiary dentin. It was reported that the cytotoxicity of SDF is related to both F and Ag constituents with a synergistic interaction between these two constituents that heightens the cellular damage 8   We assessed the effect of GSH on SDF-induced deposit formation , and the results showed that applying 20% GSH as a separate step after SDF significantly decreased the formation of these deposits with several open dentinal tubules. However, when GSH was incorporated within SDF, this inhibitory effect on deposit formation was less pronounced.
GSH was previously studied for its ability to decrease SDF-induced color changes of tooth structure. 34 The process through which GSH affects the function of SDF is still unclear; however, the interaction with Ag and/ or the acidity of GSH might have contributed to the current findings. The demineralizing effect of a low pH agent was suggested to harm the stability of crystal precipitations on dentin surfaces, 35 and this might be true for the group where 20% GSH was applied separately on dentin after SDF application. Both F and Ag ions contribute to the formation of SDF-induced precipitates. 36,37 Significant Ag layer accumulation is reported on dentin surfaces treated with SDF. 38 GSH is known to interact with silver, 19,20,39 and we speculate that the formation of these complexes could minimize the action of Ag in forming crystals on dentin surfaces.

Conclusion
The limitations of this study include, but are not