Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 µg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

PIEZO1 promotes ATP release from periodontal ligament cells following compression force.

OBJECTIVES: Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs). MATERIALS AND METHODS: To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed. RESULTS: PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1. CONCLUSIONS: PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.

2-hydroxyethyl methacrylate-derived reactive oxygen species stimulate ATP release via TRPA1 in human dental pulp cells.

Extracellular ATP (adenosine triphosphate) and transient receptor potential ankyrin 1 (TRPA1) channels are involved in calcium signaling in odontoblasts and dental pain. The resin monomer 2-hydroxyethyl methacrylate (HEMA), used in dental restorative procedures, is related to apoptotic cell death via oxidative stress. Although the TRPA1 channel is highly sensitive to reactive oxygen species (ROS), the effect of HEMA-induced ROS on ATP release to the extracellular space and the TRPA1 channel has not been clarified in human dental pulp. In this study, we investigated the extracellular ATP signaling and TRPA1 activation by HEMA-derived ROS in immortalized human dental pulp cells (hDPSC-K4DT). Among the ROS-sensitive TRP channels, TRPA1 expression was highest in undifferentiated hDPSC-K4DT cells, and its expression levels were further enhanced by osteogenic differentiation. In differentiated hDPSC-K4DT cells, 30 mM HEMA increased intracellular ROS production and ATP release, although 3 mM HEMA had no effect. Pretreatment with the free radical scavenger PBN (N-tert-butyl-α-phenylnitrone) or TRPA1 antagonist HC-030031 suppressed HEMA-induced responses. These results suggest that ROS production induced by a higher dose of HEMA activates the TRPA1 channel in human dental pulp cells, leading to ATP release. These findings may contribute to the understanding of the molecular and cellular pathogenesis of tertiary dentin formation and pain in response to dental biomaterials.

Immortalization of common marmoset-derived fibroblasts via expression of cell cycle regulators using the piggyBac transposon.

Common marmosets are non-human primate models used in biomedical research and genome editing technology. This study aimed to establish cell lines from common marmosets and evaluate their characteristics. We obtained normal fibroblasts derived from muscle tissues of two common marmosets and immortalized them with the introduction of a mutat form of cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomere reverse transcriptase (TERT) using the piggyBac transposon. Compared to parental cells, the immortalized cell lines (named K4DT cells) showed telomerase activity and an accelerated cell proliferation rate. To our knowledge, this is the first study describing the successful establishment of immortalized common marmoset-derived fibroblasts using piggyBac transposition of CDK4R24C, Cyclin D1, and TERT. Our generated cell lines might be a beneficial tool for future studies on disease modeling and targeted gene therapies.

Transcriptome analysis to identify the downstream genes of androgen receptor in dermal papilla cells.

BACKGROUND: Testosterone signaling mediates various diseases, such as androgenetic alopecia and prostate cancer. Testosterone signaling is mediated by the androgen receptor (AR). In this study, we fortuitously found that primary and immortalized dermal papilla cells suppressed AR expression, although dermal papilla cells express AR in vivo. To analyze the AR signaling pathway, we exogenously introduced the AR gene via a retrovirus into immortalized dermal papilla cells and comprehensively compared their expression profiles with and without AR expression. RESULTS: Whole-transcriptome profiling revealed that the focal adhesion pathway was mainly affected by the activation of AR signaling. In particular, we found that caveolin-1 gene expression was downregulated in AR-expressing cells, suggesting that caveolin-1 is controlled by AR. CONCLUSION: Our whole transcriptome data is critical resources for discovery of new therapeutic targets for testosterone-related diseases.

Establishment of human airway epithelial cells with doxycycline-inducible cell growth and fluorescence reporters.

We previously reported the successful establishment of multiple immortalized cell lines that preserved the original nature of the primary cells via co-expression of R24C mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). However, as these genes are kind of oncogenes, tools to control their expression levels are favorable. In this study, we describe a new polycistronic lentiviral vector expressing proliferation factors, CDK4R24C and Cyclin D1 along with enhanced green fluorescence protein (EGFP) under the control of doxycycline (Dox)-dependent transactivator (rtTA) and tetracycline response element (TRE). By introducing the Dox-inducible lentiviral vector into human airway epithelial cells, we established a novel human airway epithelial cell line harboring polycistronic Dox-inducible CDK4R24C and Cyclin D1, referred to as Tet-on K4D cells. We showed that the cell growth of Tet-on K4D cells could be controlled by Dox. Furthermore, expression of K4D genes and rtTA gene can be independently monitored by fluorescent imaging. Cultured airway epithelial cells are useful as a tool for studying the pathogenesis of lung disorders. Altogether, our established human airway epithelial cells could be used for a variety of studies such as lung pathology and biology underlying the differentiation process to form the complex pseudostratified multicellular layers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00477-0.

The transcriptome of wild-type and immortalized corneal epithelial cells.

Cellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.

Combinatorial expression of cell cycle regulators is more suitable for immortalization than oncogenic methods in dermal papilla cells.

The immortalized cell is an essential research tool that uses robust growth properties for the functional investigation of gene products. Immortalized mammalian cells have mainly been established using three methods: expression of simian vacuolating virus 40 T antigen (the SV40 method); human papilloma virus-derived oncoprotein E6/E7 (the E6/E7 method); or combinatorial expression of R24C mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase (the K4DT method). However, it is unclear as to which method is optimal for an in vitro model. Here, we compared the biological characteristics and genome-wide expression profiles of immortalized human dermal papilla cells generated by the SV40, E6/E7, or K4DT method. To our knowledge, this is the first study to comprehensively compare expression profiles to determine the optimal immortalization method for maintaining the original nature of the wild-type cells. These data would be valuable to scientists aiming to establish new immortalized cell lines.

Human Derived Immortalized Dermal Papilla Cells With a Constant Expression of Testosterone Receptor.

Androgenetic alopecia (AGA) is the most common type of hair loss, and is mainly caused by the biological effects of testosterone on dermal papilla cells (DPCs). In vitro culturing of DPCs might be a useful tool for the screening of target molecule of AGA. However, primary DPCs cannot continuously proliferate owing to cellular senescence and cell culture stress. In this study, we introduced mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) into DPCs. We confirmed protein expression of CDK4 and Cyclin D1, and enzymatic activity of TERT. Furthermore, we found the established cell line was free from cellular senescence. We also introduced the androgen receptor gene using a recombinant retrovirus, to compensate the transcriptional suppressed endogenous androgen receptor in the process of cell proliferation. Furthermore, we detected the efficient nuclear translocation of androgen receptor into the nucleus after the treatment of dihydrotestosterone, indicating the functionality of our introduced receptor. Our established cell line is a useful tool to identify the downstream signaling pathway, which activated by the testosterone.

Primary and immortalized cell lines derived from the Amami rabbit (Pentalagus furnessi) and evolutionally conserved cell cycle control with CDK4 and Cyclin D1.

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.

ADAMTSL6ß promotes fibrillin-1 microfibril assembly, which is possibly mediated via binding through the third thrombospondin type I domain to fibrillin-1.

Fibrillin-1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs-like) 6ß is one of the fibrillin-1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6ß in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6ß and examined their interactions with fibrillin-1 assembly. Pull-down assay of the ADAMTSL6ß deletion mutants and fibrillin-1 protein revealed that ADAMTSL6ß binds to fibrillin-1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin-1 matrix assembly was enhanced in MG63 cells, expressing full-length ADAMTSL6ß, when compared with that of wild type MG63 cells. While MG63 cells expressing Δ TSP3-ADAMTSL6ß form showed enhanced assembly formation, Δ TSP2-ADAMTSL6ß form did not enhance that, indicating the difference between Δ TSP2-Δ TSP3 has a critical role for fibrillin-1 assembly. As the difference of Δ TSP2-Δ TSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6ß influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6ß in the process of microfibril formation.

Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition.

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.

Fibrillin-1 insufficiency alters periodontal wound healing failure in a mouse model of Marfan syndrome.

OBJECTIVE: Marfan syndrome (MFS) is a systemic connective tissue disorder caused by insufficient fibrillin-1 (FBN-1), a major component of microfibrils that controls the elasticity and integrity of connective tissues. FBN-1 insufficiency in MFS leads to structural weakness, which causes various tissue disorders, including cardiovascular and periodontal disease. However, the role of FBN-1 insufficiency in the destruction and regeneration of connective tissue has not yet been clarified. To investigate the role of FBN-1 insufficiency in tissue destruction and regeneration. DESIGN: We used a ligature-induced (LI) periodontal disease model in fbn-1-deficient mice (fbn-1c1039G/+ mice) with MFS and investigated the regeneration level of periodontal tissue and as an inflamatic marker, the expression of the matrix metalloproteinase (mmp)-9 and tumor necrosis factor (tnf)-α. RESULTS: Interestingly, fbn-1c1039G/+ mice exhibited slowed wound healing compared with wild type mice, but periodontal tissue destruction did not differ between these mice. Moreover, fbn-1c1039G/+ mice exhibited delayed bone healing in association with continuous mmp-9 and tnf-α expression. Furthermore, inflammatory cells were obvious even after the removal of ligatures. CONCLUSION: These data suggest that fibrillin-1 insufficiency in fbn-1c1039G/+ mice interfered with wound healing in connective tissue damaged by inflammatory diseases such as periodontal disease.

F-spondin negatively regulates dental follicle differentiation through the inhibition of TGF-ß activity.

OBJECTIVE: F-spondin is an extracellular matrix (ECM) protein that belongs to the thrombospondin type I repeat superfamily and is a negative regulator of bone mass. We have previously shown that f-spondin is specifically expressed in the dental follicle (DF), which gives rise to the periodontal ligament (PDL) during the tooth root formation stage. To investigate the molecular mechanism of PDL formation, we investigated the function of f-spondin in DF differentiation. DESIGN: The expression patterning of f-spondin in the developing tooth germ was compared with that of periodontal ligament-related genes, including runx2, type I collagen and periostin, by in situ hybridization analysis. To investigate the function of f-spondin during periodontal ligament formation, an f-spondin adenovirus was infected into the bell stage of the developing tooth germ, and the effect on dental differentiation was analyzed. RESULTS: F-spondin was specifically expressed in the DF of the developing tooth germ; by contrast, type I collagen, runx2 and periostin were expressed in the DF and in the alveolar bone. F-spondin-overexpresssing tooth germ exhibited a reduction in gene expression of periostin and type I collagen in the DF. By contrast, the knockdown of f-spondin in primary DF cells increased the expression of these genes. Treatment with recombinant f-spondin protein functionally inhibited periostin expression induced by transforming growth factor-ß (TGF-ß). CONCLUSION: Our data indicated that f-spondin inhibits the differentiation of DF cells into periodontal ligament cells by inhibiting TGF-ß. These data suggested that f-spondin negatively regulates PDL differentiation which may play an important role in the immature phenotype of DF.

Metal ions from S-PRG filler have the potential to prevent periodontal disease.

The surface pre-reacted glass ionomer (S-PRG) filler, a component of composite resin, is capable of releasing metal ions that possess antibacterial activity against caries and periodontal pathogens. Although S-PRG has been suggested to be involved in oral disease prevention, no reports have been published regarding its preventive effect on periodontal disease in vivo. The present study investigated whether the eluate from S-PRG (S-PRG eluate) has a suppressive effect on tissue destruction induced in a mouse model of ligature-induced periodontal disease. Twenty-seven C57BL/6 mice were divided into three groups of nine animals each, no ligature group (Lig(-)), ligature group (Lig(+)S-PRG(-)) and ligature with S-PRG eluate group (Lig(+)S-PRG(+)). Alveolar bone loss was evaluated using micro-computed tomography scanning. Histologic changes were detected by hematoxylin and eosin staining. The infiltration of inflammatory cells was assessed by Ly6G and F4/80 staining immunohistochemically. The distribution of metal ions was detected by time-of-flight secondary ion mass spectrometry. S-PRG eluate clearly inhibited alveolar bone loss and bone density. The histological analysis revealed that S-PRG eluate reduced destruction of the collagen bundle in the periodontal ligament and the infiltration of inflammatory cells. Immunohistochemical analysis showed that the S-PRG eluate significantly suppressed the number of infiltrating neutrophils and macrophages. Time-of-flight secondary ion mass spectrometry analysis revealed that more boron ions were present in the Lig(+)S-PRG(+) group than in the Lig(+)S-PRG(-) group. Our results suggest that the S-PRG eluate has a preventive effect against tissue destruction in periodontal disease through its anti-inflammatory effects in vivo.

Structure-cytotoxicity relationship of methacrylate-based resin monomers as evaluated by an anti-oxidant responsive element-luciferase reporter assay.

To investigate the chemical structure-cytotoxicity relationship of methacrylate-based resin monomers, we studied their effects on anti-oxidant responsive element (ARE)-mediated transcription. HepG2 cells stably expressing an ARE-regulated luciferase reporter gene were cultured for 6 h with various concentrations of several resin monomers and subjected to a luciferase assay. The doseresponse curves observed for hydrophobic monomers with different hydrocarbon chains (MMA, EMA, PMA and BMA) began to rise at concentrations between 0.5 and 1 mM; the curves rose as the monomer concentrations increased up to 5 (BMA), 10 (PMA), or 30 mM (MMA and EMA). In contrast, hydrophilic monomers having a hydroxyl group (HEMA and HPMA) showed bell-shaped curves, and stimulated the reporter expression more strongly than the hydrophobic monomers in a low concentration range (0.5-5 mM). The results suggest that introduction of a hydroxyl group in a methacrylate-based resin monomer increases its intracellular electrophilic reactivity and cytotoxicity.

Isolation and characterization of the human immature osteoblast culture system from the alveolar bones of aged donors for bone regeneration therapy.

BACKGROUND: Establishment of human osteoblast cultures that retain bone-forming capacity is one of the prerequisites for successful bone regeneration therapy. Because osteoblasts harvested from adults exhibit limited growth, the use of immature osteoblasts that can expand ex vivo should greatly facilitate bone regeneration therapy. In this study, we developed immature human osteoblasts isolated from aged alveolar bone (HAOBs). METHODS: HAOBs obtained after the collagenase digestion of alveolar bones from elderly donors. Then, we assessed osteogenic ability of HAOB after treatment with recombinant human bone morphogenic protein-2 or transplantation into immunodeficient mice. In addition, we performed global gene expression analysis to identify functional marker for HAOB. RESULTS: HAOBs, which can differentiate into osteoblasts and have a robust bone-forming ability, were successfully extracted from donors who were > 60 years of age. We found that the HAOBs exhibited a higher osteogenic ability compared with those of human mesenchymal stem cells and highly expressed NEBULETTE (NEBL) with osteogenic abilities. CONCLUSIONS: HAOBs have properties similar to those of human immature osteoblasts and appear to be a novel material for cell-based bone regeneration therapy. Additionally, the expression level of NEBL may serve as a marker for the osteogenic ability of these cells.

Effect of 2-hydroxyethyl methacrylate on antioxidant responsive element-mediated transcription: a possible indication of its cytotoxicity.

BACKGROUND: The resin monomer 2-hydroxyethyl methacrylate (HEMA) is known to be more cytotoxic than methyl methacrylate (MMA). Using a luciferase reporter assay system, we previously showed that MMA activates the glutathione S-transferase alpha 1 gene (Gsta1) promoter through the anti-oxidant responsive element (ARE). However, it is not known whether HEMA induces ARE-mediated transcription. METHODOLOGY/PRINCIPAL FINDINGS: We further developed the reporter system and studied the concentration-dependent effect of HEMA on ARE enhancer activity. The revised system employed HepG2 cells stably transfected with a destabilized luciferase reporter vector carrying 2 copies of the 41-bp ARE region of Gsta1. In this system, MMA increased ARE activity by 244-fold at 30 mM; HEMA augmented ARE activity at 3 mM more intensely than MMA (36-fold versus 11-fold) and was equipotent as MMA at 10 mM (56-fold activation); however, HEMA failed to increase ARE activity at 30 mM. In HepG2 cells, HEMA detectably lowered the cellular glutathione levels at 10 mM and cell viability at 30 mM, but MMA did not. CONCLUSIONS: These results suggest that the low-concentration effect of HEMA on ARE activity reflects its cytotoxicity. Our reporter system used to examine ARE activity may be useful for evaluating cytotoxicities of resin monomers at concentrations lower than those for which cell viabilities are reduced.