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Year : 2023  |  Volume : 26  |  Issue : 3  |  Page : 275-280
Intracellular stress caused by composite resins: An in vitro study using a bioluminescent antioxidant-responsive element reporter assay

1 Department of Dental Hygiene, Aichi Gakuin University Junior College; Department of Dental Material Science, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi, Japan
2 Department of Dental Material Science, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi, Japan
3 Department of Dental Hygiene, Aichi Gakuin University Junior College, Nagoya, Aichi, Japan
4 Department of Biochemistry, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi, Japan
5 Department of Restorative Dentistry and Endodontology, Graduate School of Dentistry Osaka University, Suita, Japan

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Date of Submission11-Jan-2023
Date of Decision31-Jan-2023
Date of Acceptance23-Feb-2023
Date of Web Publication16-May-2023


Context: Elucidating the effects of leachates from composite resins (CRs) on cells by examining the transcription level of detoxification genes and the antioxidant-responsive element (ARE), would be helpful in clinical practice.
Aims: The aim of the study is to investigate the cytotoxicity of commercially available CRs, we used a reporter assay system to evaluate intracellular stress based on ARE-mediated transcription.
Setting and Design: The study design was an in vitro study.
Materials and Methods: Seven kinds of CRs were each placed in four-well plates to which culture medium was added and then light-cured. The prepared samples were used either immediately (sample A) or after incubation at 37°C for 24 h (sample B) in the subsequent ARE-luciferase reporter assay, in which HepG2 cells stably expressing an ARE-regulated luciferase reporter gene (HepG2-AD13 cells) were cultured for 6 h in culture media with the CR eluate (samples A or B) or without (control) (n = 4). In the cell viability assay, cell viability in various solutions with the same incubation time was confirmed by MTT assay (n = 4). Statistical analysis was performed using the paired t-test and one-way analysis of variance.
Results: All CR solutions showed an increase in ARE activation rate; a CR with spherical nanofillers showed the highest ARE activation rate of 108.5-fold in sample A. Cell viability was not significantly reduced for any of the CRs in sample A. However, the CR-containing bisphenol A-glycidyl methacrylate (Bis-GMA) caused a significant decrease in cell viability in sample B.
Conclusions: The intracellular stress in the viable cells differed among the CRs, depending on the type of monomer used. In particular, Bis-GMA-containing hydroxyl groups showed high cytotoxicity.

Keywords: Antioxidant-responsive element; bisphenol A-glycidyl methacrylate; composite resin; cytotoxicity; hydroxyl group; intracellular stress; luciferase reporter assay; matrix resin monomerl; MTT assay; stable clone cell

How to cite this article:
Masuda M, Hori M, Inukai J, Suzuki T, Imazato S, Kawai T. Intracellular stress caused by composite resins: An in vitro study using a bioluminescent antioxidant-responsive element reporter assay. J Conserv Dent 2023;26:275-80

How to cite this URL:
Masuda M, Hori M, Inukai J, Suzuki T, Imazato S, Kawai T. Intracellular stress caused by composite resins: An in vitro study using a bioluminescent antioxidant-responsive element reporter assay. J Conserv Dent [serial online] 2023 [cited 2023 Oct 4];26:275-80. Available from:

   Introduction Top

The main constituents of composite resin (CR) are a filler, a resin matrix, and a polymerization initiator. The resin matrix and polymerization initiator are involved in the curing reaction, but the resin matrix is not completely polymerized; the maximum polymerization rate can be as low as 60%, depending on the condition.[1],[2] Resin matrix is composed of methacrylates containing multiple double bonds, and the safety of these materials has been evaluated in multiple studies.[3],[4],[5] A previous study examining the cytotoxicity of CR has produced conflict results,[6] and the cytotoxicity varied markedly according to the leaching method used.[7] The cytotoxicity tests most frequently used include the 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazoliumbromide (MTT) assay, which judges cytotoxicity based on whether cells are dead or alive.[8],[9] Methyl methacrylate (MMA)-related molecules activate antioxidant-responsive element (ARE)-mediated transcription of detoxification genes such as GST through the Keap1-Nrf2 pathway and are detoxified through GST-catalyzed conjugation with glutathione (GSH).[10],[11] Depletion of cellular GSH has been used as a biomarker of early-stage cancer and inflammation because GSH is consumed when cells are subjected to oxidative stress or redox stimulation.[12] The detoxifying enzyme system plays an important role in determining the final fate of toxic materials and their subsequent impact on cell stress level.[13] Based on this, Orimoto et al. developed an ARE reporter assay system using a bioluminescent gene,[14] and Egashira et al. reported differences in the ARE activation rate in several acrylate monomers with different structures[15] using a stable clonal cell line (HepG2-AD13).[14] Monomers used for CR have been reported to pass through the dentin tubules and affect the pulp.[16],[17] In accordance with the concept of minimal intervention, direct pulp capping and CR filling without pulp extraction are occasionally performed in restoration cases,[18],[19] and thus, it is important to understand in detail the cytotoxicity of CRs before and immediately after polymerization.

In this study, we prepared cell culture media into which substances from CR were leached under conditions similar to those in clinical practice and investigated the level of cell stress caused by CRs. We evaluated the hypothesis that different commercially available CRs cause different levels of stress and explored the possibility of developing safer products.

   Materials and Methods Top

Composite resin specimens

The manufacturers and compositions of the tested CRs are shown in [Table 1]. [Figure 1]a shows the procedure for preparing the culture media in contact with unpolymerized CRs. First, each kind of CR was spread to a thickness of about 1 mm in a four-well plate of 15 mm in diameter. Second, culture media (Dulbecco's minimum essential medium; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) (700 μL) at 37°C was added to each well. Third, the specimens were immediately light-cured following the manufacturer's instructions using an LED curing light (Pencure 2000; Morita, Tokyo, Japan) from the bottom of the dish for 10 s, twice per well. The curing tip was placed perpendicularly in contact with the bottom of the dish. The power output density used was 1000 mW/cm2.
Figure 1: Experimental flow of ARE assay systems. (a) Preparation of samples A and B. (b) HepG2-AD13 cell stably transfected with a bioluminescent firefly luciferase gene downstream of ARE-enabled analysis of ARE-mediated gene expression by measuring luminescence. CR: Composite resin, D-MEM: Dulbecco's minimum essential medium. ARE: Antioxidant-responsive element

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Table 1: Compositions of the test materials, according to the manufacturers

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The prepared samples were used either immediately (sample A) or after incubation at 37°C for 24 h (sample B) in the subsequent ARE-luciferase report assay.

Antioxidant-responsive element-luciferase reporter assay

The steps in the workflow of the ARE-luciferase reporter assay are shown in [Figure 1]b. The HepG2-AD13 cells[14] were seeded in 24-well culture plates (Corning Inc., Corning, NY) at a density of 6.0 × 105 cells/mL (500 μL/well) and incubated for 24 h at 37°C under 5% CO2. The culture medium was then replaced with 500 μL of the fresh medium with or without the eluate for various kinds of CR (sample A or B). Cells were then incubated for 6 h and rinsed with phosphate-buffered saline (PBS) before the attachment of cells to well surfaces was confirmed under a microscope. Thereafter, cells in each well were lysed with 100 μL of Passive Lysis Buffer (Promega, Madison, WI). The plates were rotated for 15 min with a rotary shaker. The lysates were assayed for firefly luciferase activity using the Single Luciferase Reporter Assay System (Promega). Luminescence was measured in relative light units using a luminometer (AB-2200 Ver. 2.61D; ATTO, Tokyo, Japan) with an integration time of 10 s. ARE activation based on the luminescence intensity is expressed as fold activation compared with the control value obtained without the addition of the eluates. The results are expressed as the mean ± standard deviation (SD) (n = 4).

Cell viability assay

HepG2-AD13 cells were seeded in 96-well culture plates (Corning Inc.) at the same density as in the ARE assay (100 μL/well) and incubated for 24 h. The culture medium was then replaced with 100 μL of fresh medium with or without the eluate for various kinds of CRs (sample A or B). To assess cell viability after exposure to each sample for 6 h, 10 μL of WST-8 (Cell Counting Kit-8; Dojindo Molecular Technologies, Gaithersburg, MD) solution was added and incubated for 1 h. Then, the optical density of each well was measured at 450 nm with a microplate reader (Multiskan JX; Thermo Fisher Scientific, Waltham, MA). The results are expressed as the mean ± SD (n = 4).

Statistical analysis

Parametric tests were used because the ARE assays and cell viability assays had a normal distribution (Shapiro–Wilk test, P ≥ 0.05). The ARE activation relative to the control value was compared using the paired t-test for comparisons between samples A and B, whereas analysis of variance (ANOVA) was used for comparisons between CRs in sample A. For one-way ANOVA, the Levene test was used to assess unequal variances, and then the Games–Howell test was applied. The cell viability assay was compared between each CR and the control using one-way ANOVA and Dunnett's test A P < 0.05 was considered statistically significant.

Image observation

The surfaces of all specimens were sputter coated with platinum to provide electrical conductivity. Elemental analysis was performed using a field-emission electron probe microanalyzer (JXA-8530FA; JEOL, Tokyo, Japan). The probe was operated at an accelerating voltage of 10 kV and a probe current of 0.2 nA. Backscattered electron (BSE) images were acquired to obtain Z-contrast images.

   Results Top

Antioxidant-responsive element activation rate and cell viability

Microscope observation found no morphological abnormalities in cells immediately before lysis in all samples in sample A and sample B. The ARE activation rates and cell viability are shown in [Figure 2]a and [Figure 2]b, respectively. Assays were repeated multiple times and typical results are shown.
Figure 2: Results of this study. (a) ARE activity. Means followed by the same letter do not differ by the Games-Howell test. (b) Cell viability. (c) BSE images. (d) Chemical structures of resin monomers used in composite resins. *Significant different at 5% between samples A and B. ARE: Antioxidant-responsive element, BSE: Backscattered electron, Bis-GMA: Bisphenol A-glycidyl methacrylate, TEGDMA: Triethylene glycol dimethacrylate, UDMA: Urethane-dimethacrylate, Bis-MEPP: Bisphenol a ethoxylate dimethacrylate

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All samples tested showed increased ARE activation rates. In sample A, CR6 and CR7 showed the highest ARE activation rates, whereas CR1–CR5 showed significantly lower values compared with CR7 (P < 0.05), but even the lowest fold activation (by CR4) was 8.9-fold compared with the control [Figure 2]a. CR1–CR5 did not show significant differences between samples A and B, although the value for CR4 tended to be lower in the graph for sample B than in that for sample A (P = 0.068). The values for CR6 and CR7 were significantly lower in the graph for sample B than in that for sample A (P < 0.05) [Figure 2]a.

The ARE activation rate reflects cellular metabolic responses to electrophilic xenobiotics and thus depends on the number of viable cells. In sample A, the number of viable cells after stimulation did not significantly differ between any of the seven types of CR and the control, whereas in sample B, the values were significantly lower for CR3, CR4, CR6, and CR7 than for the control (P < 0.05) [Figure 2]b. Cell viability was decreased to approximately 70% compared with the control after stimulation by these CRs.

Image analysis

BSE images are shown in [Figure 2]c. Low-magnification images of CR1 and CR4 show that their structures are homogeneous, while images of CR2, CR3, and CR5 show that they contain multiple components. The filler of CR3 contained a high atomic number element, which was revealed to be ytterbium by elemental analysis. Low-magnification images show marked differences in structure between CR6 and CR7, but high-magnification images show densely packed spherical nanofiller particles of uniform size in both.

   Discussion Top

The main constituents of CR used in this study were fillers and resin matrixes. In this report, we discuss the relationship between cell stress level and the type and amount of resin matrix.

The resin matrix monomers in CR carry multiple functional groups. The distance between those functional groups is ensured by the relatively large molecular weights. The solubility of these monomers to water or culture media decreases with increasing molecular weight, but the highly polar groups in the structure affect solubility depending on the electron imbalance of the whole molecular structure.

Egashira et al. previously reported that monomers with hydroxyl groups such as hydroxyethyl methacrylate (HEMA) cause greater stress than those without hydroxyl groups such as MMA and that cell death is caused by intracellular electrophilic reactivity.[15] In the present study, bisphenol A-glycidyl methacrylate (Bis-GMA) was the only resin matrix with monomers carrying hydroxyl groups [Figure 2]d, and sample B of CRs containing Bis-GMA (CR3, CR4, CR6, and CR7) caused cell death, indicating stronger cytotoxicity in the samples. Meanwhile, although urethane-dimethacrylate (UDMA), which has an amide group [Figure 2]d, has a higher dissolution (20.4 μg/mm3) compared with Bis-GMA (9.5 μg/mm3),[20] the cell stress level of CR5 is the same as CRs with other monomers that do not have a hydroxyl group, and this cell stress does not lead to a significant decrease in cell viability. The results suggest that UDMA causes relatively lower stress on cells despite having an electron imbalance in its structure.

Next, we discuss the amount of resin matrix leached. The amounts of reactive electrophilic components leached were presumed to be similar or higher in sample B than in sample A. The lack of significant changes in ARE activation rate and cell viability in CR1, CR2, and CR5 compared with other CRs suggests that miniscule amounts of resin matrix leached from them after being cured. Triethylene glycol dimethacrylate (TEGDMA), which has a lower molecular weight compared with the other resin monomers, exhibits higher solubility (27.5 g/mm3)[20] than does Bis-GMA, but the amount of elution after curing cannot be discussed in detail in this study because the degree of polymerization of the monomer is also a key factor. However, samples composed only of bisphenol an ethoxylate dimethacrylate in CR1 and TEGDMA in CR2 showed no decrease in cell viability in sample B, and the amount eluted into the culture medium can be expected to be detoxified and metabolized. The limitations of this study are that we compared elution concentration between only two points (samples A and B), we did not examine the concentration-dependent cellular stress of each monomer, and we did not verify the amount of monomer eluted from each CR.

CR6 and CR7 showed a significantly higher ARE activation rate compared with other CRs in sample A, indicating that they caused stronger stress on cells. The dose-response curve of the ARE activation rate for HEMA was bell shaped, with a sharp drop from the peak at a certain concentration.[15] Similar bell-shaped curves would likely be observed if CR3, CR4, CR6, and CR7 were used because, similar to HEMA, they contain Bis-GMA, which contains hydroxyl groups. Taken together, the results suggest that, in sample B, the accumulated Bis-GMA that leached from samples might have caused the observed intracellular electrophilic reactivity and cytotoxicity.

   Conclusions Top

This study demonstrated the cellular detoxification responses to commercially available CRs using a luciferase reporter assay for ARE-mediated transcription. This study used a stable clone for the reporter assay, and thus the effects in terms of intracellular electrophilic stress were examined quickly and in a simple manner. Unlike conventional cytotoxicity assays, the ARE-luciferase reporter assay method can quantitatively test metabolic responses in viable cells, enabling a detailed investigation of cytotoxicity. This method is expected to contribute to the development of materials that are safer for cells.

Financial support and sponsorship

This work was supported by JSPS KAKENHI Grant Number 20K18586.

Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Dr. Miki Hori
Department of Dental Materials Science, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-Cho, Chikusa-Ku, Nagoya, Aichi 464-8650
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcd.jcd_30_23

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