Mechanism of detoxification of the cationic antibacterial monomer 12-methacryloyloxydodecylpyridiniumbromide (MDPB) by N-acetyl cysteine

Abstract

Objectives

The protective effects of N-acetyl cysteine (NAC) against cytotoxicity induced by conventional dental resin monomers have been widely documented. However, its effectiveness to detoxify cationic antibacterial monomers has not yet been elucidated. The aim of the present study was to investigate the possible protective effects of NAC against the cytotoxicity of 12-methacryloyloxydodecylpyridiniumbromide (MDPB) and explore the role of adduct formation in NAC-directed detoxification.

Methods

The influences of NAC on the cytotoxicity of MDPB were studied in mouse osteoblast-like MC3T3-E1 cells using the MTT assay. Ultra-performance liquid chromatography (UPLC) and liquid chromatography–mass spectrometry (LC–MS) analysis were performed to investigate the possible chemical reaction between NAC and MDPB.

Results

While only slight reduction in the cytotoxicity of MDPB by NAC was observed immediately after mixing with MDPB, remarkable protection against MDPB-induced cell death was detected when the mixture was tested after 24 h of pre-incubation. UPLC and LC–MS analysis revealed that chemical binding of MDPB and NAC occurred under neutral conditions after 24 h of pre-incubation.

Significance

Our findings suggest that NAC reduces the toxicity of the cationic antibacterial monomer MDPB, and adduct formation is partially responsible for the detoxification ability of NAC against MDPB-induced cell damage.

Introduction

Preservation of the pulp exposed during caries removal in permanent teeth remains one of the most controversial issues in restorative dentistry. Many clinicians often immediately resort to pulpectomy for the exposed pulp. However, because a vital pulp is important for the function of teeth, it is desirable to preserve the vitality and health of the pulp rather than replace it with a root filling material. For such a treatment strategy, capping of the pulp with the materials with antibacterial activity is considered to be of benefit.

12-Methacryloyloxydodecylpyridinium bromide (MDPB), synthesized by combining a quaternary ammonium compound with a methacrylate group , is an unique monomer incorporated in the commercial adhesive system Clearfil Protect Bond to provide antibacterial effects . Due to its sealing capacity and antibacterial effects, the MDPB-containing adhesive has been suggested to contribute to eradication of bacteria in the exposed pulp and thus create a preferable environment for the healing of pulpal tissue . However, one problem with using resin-based adhesives directly on the pulp is their irritating effects. In fact, it has been reported that pulp preservation with adhesives is occasionally associated with inflammation and necrosis of the pulp . These adverse effects can be attributed, at least partially, to toxic effects of dental resin monomers .

With attempts to enhance the biocompatibility of dental adhesives, much effort has been devoted to elucidation of the mechanisms underlying the cytotoxicity of various monomers. Although the exact and detailed mechanism is still largely unknown, many prior reports suggest that the cytotoxicity of resin monomers is related to the disturbance of intracellular redox balance . It has been noticed that the cytotoxic effects of dental resin monomers are accompanied with the depletion of the intracellular anti-oxidative enzyme glutathione (GSH) , as well as with the over-production of reactive oxygen species (ROS) .

Based on the findings that disturbance of intracellular redox balance is involved in the cytotoxic effects of resin monomers, N-acetyl cysteine (NAC), which is an anti-oxidative cysteine derivative that can be readily incorporated into cells, has been identified as an effective molecule to reduce the cytotoxicity of conventional monomers . At first, it was believed that NAC exerts protective effects against monomer-related cytotoxicity mainly through the anti-oxidative properties by directly scavenging over-produced ROS, while at the same time replenishing the exhausted intracellular GSH pool . However, very recently, some researchers have suggested an additional mechanism for the protective effects of NAC by providing evidence showing that NAC can remove dental resin monomers by directly reacting with them to form a chemical adduct .

Although the rescuing effects of NAC against conventional monomer-induced toxic effects have been well-documented, its influence on the cytotoxicity of cationic antibacterial monomers has not yet been investigated. Therefore, the purpose of the present study was to investigate the possible protection of NAC against the cytotoxicity of MDPB and explore the role of chemical binding between MDPB and NAC underlying the detoxification effects of NAC. Hence, MTT assay was performed to evaluate cell viability after treatment with MDPB alone or MDPB plus NAC. Possible chemical binding between MDPB and NAC was subsequently studied using UPLC and LC–MS.

Materials and methods

Cell culture

Mouse osteoblast-like MC3T3-E1 cells were cultured with α-minimum essential medium (α-MEM, Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, SAFC Biosciences Inc., Lenexa, KS, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen Corp.). The cells were maintained in a humidified 5% CO 2 balanced-air incubator at 37 °C.

Cell viability test

The influence of MDPB alone on the viability of MC3T3-E1 cells was studied using the MTT assay. MDPB was dissolved in complete culture medium at various concentrations ranging from 0.001 to 0.3 mM. MC3T3-E1 cells were seeded into 96-well plates at a density of 5 × 10 3 cells/well and incubated at 37 °C under 5% CO 2 for approximately 24 h. When the cells grew to 80% confluence, the medium was replaced by one containing MDPB and the cells were cultured for 24 h. Then, 10 μL of MTT solution (5 mg/mL, Sigma–Aldrich, St. Louis, MO, USA), disinfected by infiltration, was added to each well and the cells were incubated for a further 4 h. After disposal of the culture medium and addition of 200 μL dimethyl sulfoxide (Amresco, Solon, OH, USA), the absorbance of each well at 492 nm was measured. For the blank group, complete culture medium without cells was inoculated. For the control group, cells were treated with complete culture medium without MDPB. The experiments were repeated three times with five replicates, and the relative viability of the cells was calculated using the following equation:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='Relative cell viability=ODexperimental−ODblankODcontrol−ODblank’>Relative cell viability=ODexperimentalODblankODcontrolODblankRelative cell viability=ODexperimental−ODblankODcontrol−ODblank
Relative cell viability = OD experimental − OD blank OD control − OD blank

The MTT assay was also employed to evaluate the influence of NAC on the cytotoxicity of MDPB. First, the influence of NAC alone (1, 5, 10, 15 and 20 mM, pH adjusted to 7.2 with 0.1 M of NaOH) on the viability of the cells was studied. Then, to evaluate the effects of NAC (1, 5 and 10 mM) on MDPB-induced cell death, the mixture of NAC and MDPB was prepared and used to treat cells either immediately after mixing or after 24 h of pre-incubation at 37 °C. The MTT assay was performed as described above.

Analysis of MDPB–NAC adduct formation with ultra-performance liquid chromatography (UPLC)

MDPB and NAC were mixed in three ways as shown in Table 1 , and the formation of the MDPB–NAC adduct was investigated. In each group, the mixture of MDPB and NAC was analyzed either immediately after preparation or after 24 h of pre-incubation. The detection was performed at 220 nm on a 1.7 μm ACQUITY UPLC C18BEH column (2.1 mm × 100 mm, Waters, Milford, MA, USA) using an ACQUITY UPLC system (Waters). The column was maintained at 50 °C and the samples were eluted for 10 min. The mobile phase, at a flow rate of 0.4 mL/min, consisted of methanol and 0.2% phosphoric acid with 100 mM sodium perchlorate. The volume of sample injected was 10 μL. The analysis was repeated three times for each group.

Table 1
MDPB/NAC mixture preparation for UPLC study.
Groups Preparation method
Group 1: NAC + MDPB (pH = 2.7) 10 mM NAC solution (in water) was prepared and pure MDPB was added directly to this solution to a final concentration of 8 mM. The final pH was 2.7
Group 2: NAC + MDPB + NH 3 (pH = 7.0) 10 mM NAC solution (in water) was first neutralized with 0.3% NH 3 and then pure MDPB was added to a final concentration of 8 mM. The final pH was 7.0
Group 3: NAC + MDPB + NaOH (pH = 7.0) 10 mM NAC solution (in water) was first neutralized with 0.1 M NaOH and then pure MDPB was added to a final concentration of 8 mM. The final pH was 7.0

Analysis of MDPB–NAC adduct with liquid chromatography–mass spectrometry (LC–MS)

The reaction mixture of NAC and MDPB was further analyzed using LC–MS to confirm the formation of the MDPB–NAC adduct. NAC aq. solution at 10 mM was neutralized with 0.3% NH 3 and pure MDPB was added to a final concentration of 8 mM. The mixture was incubated for 16 h to allow the reaction occur, and then LC–MS analysis was performed using a LCT-Premier system (Waters). Different components in the reaction mixture were separated with a TSK gel 80Ts C18 column (4.6 mm × 150 mm, TOSOH, Tokyo, Japan) at a flow rate of 1 mL/min. The column was maintained at 40 °C and the mobile phase consisted of 5 mM trifluoroacetic acid and methanol. The eluents were introduced into the mass spectrometer by electrospray ionization, with capillary and cone voltages set in the positive mode to 2400 and 30 V. Data were acquired in MS scanning mode and recorded in the 100–1000 m / z region. The analysis was repeated three times for each group.

Statistical analysis

The results of the MTT assay were statistically analyzed using one-way ANOVA and post hoc Dunnett test at a significance level of 0.05.

Materials and methods

Cell culture

Mouse osteoblast-like MC3T3-E1 cells were cultured with α-minimum essential medium (α-MEM, Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, SAFC Biosciences Inc., Lenexa, KS, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen Corp.). The cells were maintained in a humidified 5% CO 2 balanced-air incubator at 37 °C.

Cell viability test

The influence of MDPB alone on the viability of MC3T3-E1 cells was studied using the MTT assay. MDPB was dissolved in complete culture medium at various concentrations ranging from 0.001 to 0.3 mM. MC3T3-E1 cells were seeded into 96-well plates at a density of 5 × 10 3 cells/well and incubated at 37 °C under 5% CO 2 for approximately 24 h. When the cells grew to 80% confluence, the medium was replaced by one containing MDPB and the cells were cultured for 24 h. Then, 10 μL of MTT solution (5 mg/mL, Sigma–Aldrich, St. Louis, MO, USA), disinfected by infiltration, was added to each well and the cells were incubated for a further 4 h. After disposal of the culture medium and addition of 200 μL dimethyl sulfoxide (Amresco, Solon, OH, USA), the absorbance of each well at 492 nm was measured. For the blank group, complete culture medium without cells was inoculated. For the control group, cells were treated with complete culture medium without MDPB. The experiments were repeated three times with five replicates, and the relative viability of the cells was calculated using the following equation:

Relative cell viability=ODexperimentalODblankODcontrolODblankRelative cell viability=ODexperimentalODblankODcontrolODblank
Relative cell viability = OD experimental − OD blank OD control − OD blank
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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Mechanism of detoxification of the cationic antibacterial monomer 12-methacryloyloxydodecylpyridiniumbromide (MDPB) by N-acetyl cysteine
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