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ORIGINAL ARTICLE
Year : 2017  |  Volume : 20  |  Issue : 11  |  Page : 1368-1403

Microarray analysis of the gene expression profile in triethylene glycol dimethacrylate-treated human dental pulp cells


1 Department of Medical Genetics, Gulhane Military Medical Academy, Ankara 06018, Turkey
2 Department of Restorative Dentistry and Endodontics, Gulhane Military Medical Academy, Ankara 06018, Turkey
3 Department of Medical and Cancer Research Center, Gulhane Military Medical Academy, Ankara 06018, Turkey
4 Department of Medical and Cancer Research Center; Department of Haematology, Gulhane Military Medical Academy, Ankara 06018, Turkey

Date of Acceptance06-Apr-2016
Date of Web Publication05-Jan-2018

Correspondence Address:
Dr. Z Ö Torun
Department of Restorative Dentistry and Endodontics, Gulhane Military Medical Academy, Etlik, Ankara 06018
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1119-3077.181353

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   Abstract 


Objective: Triethylene glycol dimethacrylate (TEGDMA) is an important resin monomer commonly used in the structure of dental restorative materials. Recent studies have shown that unpolymerized resin monomers may be released into the oral environment and cause harmful biological effects. We investigated changes in the gene expression profiles of TEGDMA-treated human dental pulp cells (hDPCs) following short- (1-day) and long-term (7-days) exposure. Materials and Methods: HDPCs were exposed to a noncytotoxic concentration of TEGDMA, and gene expression profiles were evaluated by microarray analysis. The results were confirmed by quantitative reverse-transcriptase PCR (qRT PCR). Results: In total, 1282 and 1319 genes (up- or down-regulated) were differentially expressed compared with control group after the 1- and 7-day incubation periods, respectively. Biological ontology-based analyses revealed that metabolic, cellular, and developmental processes constituted the largest groups of biological functional processes. qRT-PCR analysis on bone morphogenetic protein-2 (BMP-2), BMP-4, secreted protein, acidic, cysteine-rich, collagen type I alpha 1, oxidative stress-induced growth inhibitor 1, MMP3, interleukin-6, and heme oxygenase-1 genes confirmed the changes in expression observed in the microarray analysis. Conclusions: Our results suggest that TEGDMA can change the many functions of hDPCs through large changes in gene expression levels and complex interactions with different signaling pathways.

Keywords: Gene expression, human dental pulp cell, microarray, triethylene glycol dimethacrylate


How to cite this article:
Torun D, Torun Z &, Demirkaya K, Sarper M, Elçi M P, Avcu F. Microarray analysis of the gene expression profile in triethylene glycol dimethacrylate-treated human dental pulp cells. Niger J Clin Pract 2017;20:1368-403

How to cite this URL:
Torun D, Torun Z &, Demirkaya K, Sarper M, Elçi M P, Avcu F. Microarray analysis of the gene expression profile in triethylene glycol dimethacrylate-treated human dental pulp cells. Niger J Clin Pract [serial online] 2017 [cited 2018 Apr 22];20:1368-403. Available from: http://www.njcponline.com/text.asp?2017/20/11/1368/181353




   Introduction Top


Resin monomers are widely used in dentin bonding agents and composite resins to restore teeth structures impaired by caries or fractures. With its hydrophilic structure and low molecular weight, triethylene glycol dimethacrylate (TEGDMA) is an important resin monomer and undergoes rapid polymerization after light curing. Due to its hydrophilic nature, the degradation processes and insufficient polymerization of TEGDMA cause the release of dental resin monomers into the oral environment, which can trigger hazardous biological effects on living oral tissues.[1],[2] Dentin thickness and the severity of the caries lesion are important factors in determining the amount of resin monomers interacting with dental pulp tissue.[3]

Various studies have been performed to show the adverse biological effects of TEGDMA on different mammalian cells. Previous studies revealed that TEGDMA has considerable cytotoxicity against different cell types via DNA damage, caspase activation, induction of apoptotic proteins, and reactive oxygen species.[4],[5],[6],[7],[8],[9],[10] TEGDMA also influences the odontogenic differentiation capacity of dental pulp cells by decreasing the expression of mineralization-related genes.[11] TEGDMA can cause changes in the immune system by affecting cytokine production and the expression of surface markers that are essential for immune cells.[12],[13] These findings suggest that TEGDMA interacts with living cells by influencing different biological pathways and causing adverse effects.

Recently, high-throughput procedures such as DNA microarray and RNA-seq technologies have been used to investigate the effects of high and low doses of TEGDMA on skin fibroblasts and human dental pulp cells (hDPCs), respectively.[9],[10],[11] These studies have greatly contributed to scientific knowledge regarding the basic mechanisms of action of TEGDMA, but unknown issues remain to be determined. Microarray analysis can be used to decipher the expression state of tens of thousands of genes in a single experiment. Microarrays provide an opportunity to explore the effects of various materials and allow researchers to evaluate the overall state of a cell. In addition, gene ontology databases contribute to the understanding of predominant biological processes, pathways, and functional regulatory networks from the microarray data.

In the present study, we tested the hypothesis that an increase in the exposure time of hDPCs to TEGDMA may differentially alter the gene expression profile of hDPCs. Thus, hDPCs and high-throughput DNA microarray analysis were used to test whether TEGDMA changed gene expression profiles after 1- and 7-day exposure periods.


   Materials and Methods Top


Cell culture

This study was approved by the local Ethics Committee. Dental pulp tissues were obtained from the molars of healthy patients undergoing orthodontic treatments. Extracted molars were kept in phosphate-buffered saline solution (Biological Industries, Kibbutz Beit Haemek, Israel) containing 100 U/mL penicillin and 100 μg/mL streptomycin (Biological Industries) to eliminate bacterial contamination. After transferring to the laboratory, extracted molars were cut horizontally 1 mm below the cementoenamel junction. The pulp tissues were gently separated from the crown and root and placed in a 100-mm  Petri dish More Details. Pulp tissues were cut into small pieces with a blade and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Biological Industries) containing 10% fetal bovine serum (FBS; Biological Industries), 100 U/mL penicillin, and 100 μg/mL streptomycin (Biological Industries). Tissue cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C. Cells from the fifth passage were used for subsequent experiments and cultured for 24 h before analysis.

XTT assay

The XTT assay was used to determine the noncytotoxic dose of TEGDMA. The XTT assay is useful for determining cellular proliferation and viability by spectrophotometric quantification. The assay is used to measure cell proliferation in response to growth factors, cytokines, and nutrients based on the conversion of the yellow tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) to an orange formazan dye by metabolically active and viable cells. Cells (100 μL/well) were seeded into 96-well plates at 2 × 104 per well and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37°C. Then, three groups were prepared: 0.3 mm TEGDMA, 1 mm TEGDMA, and 3 mm TEGDMA. TEGDMA was purchased from Sigma-Aldrich (Sigma Chemical Company, St. Louis, MO, USA). Untreated cell cultures were used as control. The cell cultures were exposed to serial dilutions of the test materials. After an incubation period of 7 days, 50 μL (0.3 mg/mL) of XTT labeling mixture (Cell Proliferation Kit II; Roche, Mannheim, Germany) was added to each well, followed by incubation for 4 h in a humidified atmosphere of 5% CO2 at 37°C. The absorbances of the metabolized media were measured at 450 nm using a microplate reader (ELX800BKT, Bio-Tek Instruments, USA). Cell viabilities of the test groups were calculated as a percentage of the control group. Each experimental group consisted of nine samples.

Microarray analysis

The TEGDMA concentration to be used for microarray analysis was decided by determining where it exhibited significantly different (P< 0.05) but higher cell viability (>90%) after 7 days. Thus, 1 mm TEGDMA was selected as the experimental dosage. hDPCs were seeded into 12-well plates at 1 × 106 per well and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37°C. Cells were pooled 1; 7 days after, 1 mm TEGDMA was applied. Cell pooling was conducted from 3 wells of each group. Wells without TEGDMA were also cultured for 1 and 7 days, and untreated hDPCs were used as controls. Each experimental group consisted of four samples.

Total RNA from hDPCs was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA purity and integrity were quantified using a p360 Nanophotometer (Implen, Germany). The microarray analysis was performed using the GeneChip 3 IVT Express Kit (Affymetrix, USA). Briefly, total RNA was subjected to reverse transcription ( first-strand cDNA synthesis), converted into double-strand cDNA,in vitro transcription, purification, and fragmentation. The samples were hybridized onto the GeneChip PrimeView Human Gene Expression Array (Affymetrix), which covers more than 36,000 transcripts and variants. After hybridization for 16 h at 45°C, the arrays were washed and then scanned to obtain quantitative gene expression levels.

Data were analyzed using the Expression Console and Transcriptome Analysis Console/Partek Genomic Suite. Raw data were normalized by the robust multiarray average algorithm. Array data were filtered by detection P < 0.05. A comparative analysis between each sample was carried out using fold-change data. Biological, ontology-based analyses were conducted using the PANTHER database (http://www.pantherdb.org).

Quantitative real-time PCR (qRT-PCR) analysis

To confirm the results obtained from the microarray experiments, qRT-PCR was performed. Bone morphogenetic protein-2 (BMP-2), BMP-4, secreted protein, acidic, cysteine-rich (SPARC), collagen type I alpha 1 (COL1A1), oxidative stress-induced growth inhibitor 1 (OSGIN1), matrix metallopeptidase-3 (MMP-3), interleukin-6 (IL-6), and heme oxygenase-1 (HMOX1) genes were chosen due to their relationship with mineralization, bone formation, extracellular matrix (ECM) formation, DNA damage, oxidative stress, apoptosis, and inflammation, respectively. β-actin (ACTB) was used as a housekeeping gene to normalize RNA expression. qRT-PCR analyses were conducted using the total RNA samples previously described for the microarray analyses. cDNA was synthesized from 25 ng of total RNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). The cDNA obtained was used as a template for PCR. The target cDNA was then amplified using specific primer pairs [Table 1]. qRT-PCR was performed using the Faststart Essential DNA Green Master (Roche) and a LightCycler Nano Instrument (Roche).
Table 1: Primer sequence list in qRT-PCR

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The 20-μL reaction mixture consisted of 5 μL cDNA, 4 μL water, 10 μL × 2 master mix buffer, and a final concentration of 0.25 pmol/μL of each primer. qRT-PCR conditions included an initial denaturation step at 95°C for 10 min, followed by 45 cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The mRNA level in each sample was calculated using ΔΔCT (i.e., ΔCT [treated sample] −ΔCT [untreated sample]) method. Each experiment was performed in triplicate.

Statistical analysis

SPSS software (version 21.0; IBM, Chicago, IL, USA) was used for all calculations. The distributions of all numerical variables, including BMP-2, BMP-4, SPARC, COL1A1, OSGIN-1, MMP-3, IL-6, and HMOX1 mRNA levels, were skewed; thus, results were reported as medians and interquartile ranges. The Mann–Whitney U-test was used to compare numerical variables between groups. A P< 0.05 was considered statistically significant.


   Results Top


Microarray data analyses

Only genes showing expression changes >2-fold after 1 mm TEGDMA treatment were taken into consideration. In total, 1282 and 1319 genes (up- or down-regulated) were differentially expressed compared with control group after 1- and 7-day incubationw periods, respectively. Of these, 276 genes were upregulated and 521 were downregulated in both time periods. Also, 481 and 518 genes exhibited statistically significant differential expression (up- or down-regulated) solely after 1- or 7-day incubation period, respectively. Transcripts with altered expression levels and biological, ontology-based analyses are reported in [Supplementary Table 1] and [Supplementary Table 2].

The largest groups of upregulated genes were involved in metabolic processes (GO: 0008152), cellular processes (GO: 0009987), developmental processes (GO: 0032502), localization (GO: 0051179), biological regulation (GO: 0065007), responses to stimuli (GO: 0050896), immune system processes (GO: 0002376), cellular component organization or biogenesis (GO: 0071840), biological adhesion (GO: 0022610), apoptotic processes (GO: 0006915), reproduction (GO: 0000003), and growth (GO: 0040007) [Figure 1]. The largest groups of downregulated genes were involved in metabolic processes (GO: 0008152), cellular processes (GO: 0009987), developmental processes (GO: 0032502), biological regulation (GO: 0065007), multicellular organismal processes (GO: 0032501), immune system processes (GO: 0002376), localization (GO: 0051179), responses to stimuli (GO: 0050896), biological adhesion (GO: 0022610), cellular component organization or biogenesis (GO: 0071840), apoptotic processes (GO: 0006915), reproduction (GO: 0000003), locomotion (GO: 0040011), and growth (GO: 0040007) [Figure 2].
Figure 1: Diagram of the largest groups of upregulated biological processes in triethylene glycol dimethacrylate-treated human dental pulp cells after (a) 1- and (b) 7-day incubation period. The numbers designated in the pieces represent the number of genes those appear in any given category

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Figure 2: Diagram of the largest groups of downregulated biological processes in triethylene glycol dimethacrylate-treated human dental pulp cells after (a) 1- and (b) 7-day incubation period. The numbers designated in the pieces represent the number of genes those appear in any given category

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The genes that showed expression changes >10-fold are listed in [Table 2]. The THBD gene exhibited the highest expression level, with 88.54- and 57.65-fold changes after 1- and 7-day incubation periods, respectively. The COL1A1 and FDNC1 genes showed the most dramatic downregulation, with −57.48- and −128.02-fold changes, respectively.
Table 2: Genes those showed up- and down-regulation of more than 10-fold changes after 1 mm triethylene glycol dimethacrylate treatment

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qRT-PCR validation

The microarray data were validated using qRT-PCR on the following genes: BMP-2, BMP-4, SPARC, COL1A1, OSGIN-1, MMP-3, IL-6, and HMOX1. These eight genes are related to mineralization, bone formation, ECM formation, DNA damage, oxidative stress, apoptosis, and inflammation. qRT-PCR results are shown in [Figure 3]. The qRT-PCR results confirmed the changes in expression revealed by the microarray analysis. hDPCs treated with 1 mm TEGDMA showed increased expression levels of BMP-2, OSGIN-1, MMP-3, and HMOX1, and decreased expression of BMP-4, SPARC, COL1A1, and IL-6 (all P < 0.05) compared with control group.
Figure 3: Verification of the microarray data using qRT-PCR on the following genes: (a) Bone morphogenetic protein-2, bone morphogenetic protein-4, secreted protein, acidic, cysteine-rich, collagen type I alpha 1, interleukin-6, oxidative stress-induced growth inhibitor 1, (b) matrix metalloproteinase 3, and heme oxygenase-1. Statistically significant differences are indicated by asterisks (P < 0.05)

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   Discussion Top


Resin monomers are used quite widely in dental practice. However, monomers cause many adverse biological effects in exposed cells and disturbance in many regulatory cellular mechanisms in cells interacting with some of these monomers. Previous studies have used high-throughput technologies such as DNA microarray and RNA-seq to investigate the biological effects of TEGDMA on skin fibroblasts and hDPCs, respectively.[9],[11] These studies revealed that TEGDMA leads to considerable gene expression changes over time and affects many different signaling pathways. However, the effects of TEGDMA interacting with hDPCs over a relatively long time period are not understood. Knowledge of the cellular mechanisms associated with the adverse effects of TEGDMA will provide a better understanding to improve materials being used and may lead to innovative therapeutic strategies. In this study, we sought to determine the effects of TEGDMA on hDPCs after a 7-day exposure period. The results of this study confirmed a highly variable gene expression profile of hDPCs after exposure to TEGDMA and the capacity of TEGDMA to induce DNA damage, oxidative stress, apoptosis, inflammation, and negative regulation of dentin mineralization.

Metabolic, cellular, and developmental processes were the most affected biological functional annotations in both up- and down-regulated transcripts. However, cell-cell signaling, cell-cell adhesion, cell cycle, induction of apoptosis, cell death, macrophage activation, ion transport, and responses to stress were the most affected subgroups of biological processes [Appendix Table 1] and [Appendix Table 2]. The competence of cells to sense and respond to their microenvironment is the basis of development, tissue repair, and immunity, as well as normal tissue homeostasis. These results reflect that the genes showing the greatest changes in hDPCs exposed to TEGDMA were associated primarily with basic cellular activities, such as inflammation and wound healing, and that TEGDMA causes changes in the coordination of cell actions. TEGDMA also caused some unusual systemic biological effects associated with fertilization and gamete generation. hDPCs are localized in an environment that is surrounded by hard dentin tissue, and nutritional support for hDPCs comes only through vessels in root canals. Thus, investigation of the release of unpolymerized dental resin monomers into the systemic circulation and possible effects on fertility is an important research topic.[14],[15]



In addition, the results of the present study revealed important findings regarding the influence of TEGDMA on the mineralization of pulp cells. The genes most associated with pulp mineralization in this study were BMP-2, BMP-4, SPARC, and COL1A1. These genes encode ECM proteins and are used as markers of odontoblastic differentiation.[16],[17],[18] TEGDMA exhibited both positive and negative regulation on the expression of mineralization-related genes. While hDPCs showed an increase in BMP-2 expression after exposure to TEGDMA, BMP-4, SPARC, and COL1A1 were decreased in a time-dependent manner. The results of the present study are consistent with a recent study that concluded that the dose of TEGDMA applied, and the influence of TEGDMA on the different intracellular signaling pathways might affect the mineralization of pulp cells in different ways.[11] In addition, Galler et al. showed that TEGDMA caused dose- and time-dependent decreases in the expression of genes associated with pulp mineralization, including collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein.[3] Moreover, our results provide insights into other mineralization-related genes and have expanded the effects of TEGDMA on dentin mineralization.

Wound healing is a complex process in which hemostasis, inflammation, angiogenesis, ECM formation, remodeling by cell death, and apoptosis constitute some of the important stages in this process.[19] The results of the present study provide significant data regarding the effects of TEGDMA on tissue repair. hDPCs exposed to TEGDMA showed up- and down-regulation in genes mostly associated with responses to oxidative stress and inflammation. In addition, angiogenesis-, blood coagulation-, proliferation-, and differentiation-related genes were also identified, which changed significantly over time [Appendix Table 1] and [Appendix Table 2]. OSGIN-1, oxidative stress-induced growth inhibitor 1, encodes an oxidative stress response protein and regulates apoptosis.[20] It also appears to be a key regulator of both inflammatory and anti-inflammatory molecules. HMOX1 plays a key role in the metabolism of heme and acts as a protective mechanism in the presence of oxidative stress.[21],[22] In the present study, TEGDMA-exposed hDPCs exhibited upregulated expression of the OSGIN-1 gene after 1- and 7-day exposure periods compared with control group. However, TEGDMA caused a greater fold change only after 1-day incubation whereas a significant expression change after 7-day incubation period was not detected. This might reflect the massive expression of HMOX1 after the early exposure stage of hDPCs as an indicator of the initial protective effect of HMOX1 against TEGDMA-induced reactive oxygen species, and other genes, such as OSGIN-1, continue to function as a part of the oxidative stress-induced biological pathways. Previous studies have already shown initial HMOX1 expression in TEGDMA-treated hDPCs.[11] However, OSGIN-1 and associated signaling pathways seem to be an attractive research area to examine the TEGDMA-hDPCs interaction in terms of responses to oxidative stress.

Matrix metallopeptidase (MMP) are calcium- and zinc-dependent endopeptidases that play roles in the remodeling and degradation of ECM.[23] However, the role of MMPs in wound healing is unclear. While Hiyama et al. reported that MMP-3 promotes and accelerates wound healing, Beidler et al. and Liu et al. reported that elevated MMP levels are associated with nonhealing.[24],[25],[26]MMP-3 was upregulated after 1- and 7-day exposure times in the present study. This suggests that MMP-3 may be involved in the wound healing process of pulp tissue treated with TEGDMA via reorganization of the ECM. However, further studies are needed to establish whether MMP-3 increases or reduces wound healing in TEGDMA-treated hDPCs.

IL-6 acts as a pro- and anti-inflammatory cytokine, and the release of this cytokine is important in the initiation of inflammatory processes after exposure to harmful foreign pathogens, such as viruses, bacteria, and chemicals.[13] Various studies have been carried out on different cell lines to show the effects of resin monomers on IL-6 sec retion, and different results were obtained regarding the responses of the cells. A TEGDMA-treated three-dimensional human tissue model constructed from TR146 cells revealed an increase in the secretion of IL-6.[27] It has also been reported that costimulation of macrophages with lipopolysaccharides and TEGDMA resulted in a dose-dependent decrease in the secretion of IL-6.[13] However, no significant change was detected in IL- 6 production after the macrophages were treated with increasing concentrations of TEGDMA alone. In the present study, IL-6 expression in TEGDMA-exposed hDPCs was inhibited after 1-day incubation period. However, we did not detect a significant expression change after 7-day incubation period. Although there are some contradictions, current and previous results described here reveal that TEGDMA has an important effect on inflammatory processes in terms of its inhibition and/or stimulation. Experimental conditions and differences in the cell lines used may be a cause of the differences. However, the results presented herein suggest that TEGDMA inhibits the innate immune system in hDPCs by inhibiting the production of cytokines, including IL-6. Disturbances of the innate immune system in the presence of TEGDMA may prevent hDPCs from generating a proper immune response and make the host vulnerable to pathogens.

Taken together, these findings revealed that various biological pathways contribute to the adverse effects of TEGDMA on hDPCs. Physical properties, such as low molecular weight and relatively high hydrophilicity, are the important factors for the penetration of TEGDMA to all biological compartments, and this may be the cause of different chemical-biological interactions with intracellular processes.[28] The present study revealed that the vast majority of the differentially regulated transcripts play a role in DNA damage, oxidative stress, apoptosis, and negative regulation of dentin mineralization. Besides, TEGDMA revealed important changes in the inflammation and wound healing processes. Biocompatibility is defined as the absence of inflammatory, irritating, toxic, or genotoxic effects in biological systems and the aforementioned data revealed that TEGDMA does not have most of these biocompatibility features and cause multiple adverse effects on interacting cells. Consequently, it should be the aim of future studies to show the effects of TEGDMA in detail about interactions with intracellular processes and to develop more biocompatible monomers.

The small number of samples analyzed is a limitation of this study. However, the data provide new information about the gene expression profiles of hDPCs in response to TEGDMA treatment.


   Conclusions Top


While the detailed mechanism of toxicity is not completely understood, the present study revealed that TEGDMA can change the many functions of hDPCs through large changes in gene expression levels and complex interactions with different signaling pathways.

Financial support and sponsorship

This study was supported by the Gulhane Military Medical Academy Research and Development Center, Ankara/Turkey (No: AR-2012/11).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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