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Components of Dentinal Adhesives Modulate Heat Shock Protein 72 Expression in Heat-stressed THP-1 Human Monocytes at Sublethal Concentrations
1 Department of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan; Correspondence: *corresponding author, nodam{at}den.hokudai.ac.jp
Few studies have investigated the ability of dental resins to induce cellular stress at sublethal concentrations. Cellular stress, especially in immune cells such as monocytes, may modulate the biological response to materials or the host's ability to respond to bacterially mediated inflammation. The current study examined the ability of sublethal concentrations of 2-hydroxylethylmethacrylate (HEMA) and triethyleneglycol dimethacrylate (TEGDMA) to induce heat shock protein 72 (HSP72) in human monocytes. HEMA and TEGDMA significantly suppressed heat-induced HSP72 expression, even at sublethal levels, but did not induce HSP72 by themselves. The results of the current study suggest that components released from dental resin could modulate the HSP stress response without altering cellular metabolic activity.
Key Words: dental adhesive • monomers • cell stress
Dentin bonding agents release components, especially 2-hydroyethylmethacrylate (HEMA) and triethyleneglylcol dimethacryate (TEGDMA), after polymerization. This release is highly dependent on the degree of polymerization of the system, which is never complete (Ferracane, 1994; Gerzina and Hume, 1996; Geurtsen et al., 1998, 1999). Clinically, the popularity of resin-based dentinal adhesives has widened their use. Dentinal adhesives are now routinely used in deep preparations over large areas of dentin, or even as direct pulp-capping agents (Costa et al., 2002; Schuurs et al., 2000). Thus, the pulpal tissues may be exposed to high concentrations of these compounds. Several reports have confirmed the dentinal diffusion of HEMA and TEGDMA in sufficient concentrations to cause cellular damage (Bouillaguet et al., 1996, 1998; Gerzina and Hume, 1996). Other reports have shown significant cellular metabolic effects of HEMA and TEGDMA at subtoxic concentrations during extended exposures of 2-5 wks (Bouillaguet et al., 2000; Noda et al., 2002). Longer-term degradation of polymerized resins at the dentin-resin interface may also contribute to pulpal exposure (Tanaka et al., 1999; Hashimoto et al., 2000). The biocompatibility of dentinal adhesives is related to resin component release and has been assessed in vitro (Camps et al., 1997; Yoshii, 1997; Bouillaguet et al., 1998, 2000; Geurtsen et al., 1998). Investigators have measured cellular growth, death, mitochondrial activity, protein synthesis, or other basic cellular metabolic activities as indicators of cellular response to resin components. However, the intracellular mechanisms altered by resin components are still not clear. Intra-orally, cells are exposed to many stressors from materials or bacteria, or from mechanical or thermal sources. Heat shock proteins (HSPs) are multifunctional proteins known to be expressed where cells are exposed to stressors including elevated temperatures, chemicals, or bacterial products. HSPs play a key role as molecular chaperones (Bachelet et al., 1998; Fink, 1999; Yenari et al., 1999). They contribute to the correct folding of proteins that have lost or not yet acquired their normal three-dimensional structure. HSPs therefore help to protect cells against stress. HSP72 is a member of the HSP family. HSP72 is known as a stress-induced protein in mammalian cells and is therefore a logical indicator of cellular stress (Ohshima et al., 1997). There is little information about the ability of resins to modulate the expression of HSPs. We hypothesized that if material components stress cells, this stress may be manifest by induction or modulation of HSP72 expression. To investigate this hypothesis, we exposed monocytes to HEMA or TEGDMA, major components of dentinal adhesives. The monocytes were considered a logical target because these cells orchestrate and amplify the inflammatory response to biomaterials (Auger and Ross, 1992).
Cell Culture Human THP-1 monocytes (ATCC TIB 202, American Type Culture Collection, Rockville, MD, USA) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 mmol/L of glutamine, 100 units/mL of penicillin, 100 µg/mL streptomycin (Gibco, BRL, Gaithersburg, MD, USA), and 50 µmol/L of β-mercaptoethanol (Sigma, St. Louis, MO, USA), and incubated at 37°C and 5% CO2 in air.
Monomers and Concentrations To determine concentrations of resins for the HSP experiments, we incubated THP-1 cells with HEMA (0-40 mmol/L) or TEGDMA (0-3 mmol/L) for 2, 24, or 48 hrs, and measured succinic dehydrogenase (SDH) activity by the MTT assay (Wataha et al., 1992). All reagents used in the current experiment were chemical grade. After incubation with HEMA or TEGDMA, the cells (100,000 cells/mL, n = 8 for each time and concentration) were plated in 96-well plates. MTT (Sigma) solution was added at 1 mg/mL for 1 hr, after which the solution was removed. The cells were fixed with Tris-formalin (0.2 mol/L of Tris, 4% v/v formalin) and washed with double-distilled water. Finally, a dimethylsulfoxide solution (6.25% v/v DMSO in 0.1 mol/L NaOH) was added to dissolve the formazan, and the resulting solution was read in a spectrophotometer at 562 nm. Concentrations for the subsequent HSP experiments were chosen to be at or below toxic concentrations at 24 hrs. The concentrations used for HSP experiments were therefore 0, 2.5, 5, and 10 mmol/L for HEMA, and 0, 0.25, 0.5, and 1.0 mmol/L for TEGDMA.
Conditions of Heat Shock Stress and Recovery To measure HSP expression, we first added HEMA or TEGDMA to THP-1 cultures (5.0 x 106 cells/5 mL, n = 6), then applied 1 hr of heat stress. We allowed the culture to recover for 2, 4, or 6 hrs at 37°C so that there would be adequate time for HSP expression to occur (Polla et al., 1995; Bachelet et al., 1998; Guzik et al., 1999). Control groups received no heat stress, but remained at 37°C for an equivalent time (1 hr sham stress plus 6 hrs recovery). Cells were processed for SDS-PAGE and Western blotting for the measurement of HSP expression.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Immunoblotting
HEMA and TEGDMA had little effect on the SDH activity of the THP-1 monocytes (Fig. 1  ). In the absence of heat stress, HEMA and TEGDMA had no significant effects on the SDH activity during the seven-hour exposure (ANOVA, p = 0.926 and 0.739, respectively). When 1 hr of heat stress was applied, there were no significant differences in SDH activity of the control cultures (without resins, Fig. 1, y axes, open vs. solid circles). For HEMA and TEGDMA, the combination of heat plus resins significantly depressed SDH activity about 15% at the highest concentrations only (vs. heat-stressed controls with no resin). At the highest concentrations, the depression of SDH activity was also significant with respect to unstressed, unexposed controls (two-tailed t test, p = 10-10 for HEMA, 10-7 for TEGDMA).
Figs. 2 and 3 show the effects of HEMA and TEGDMA on HSP72 expression in THP-1 (Fig. 2 is a typical SDS-PAGE and Western blot). There was no indication that HEMA alone (without heat stress) caused HSP72 expression (Fig. 3). However, the data clearly showed a significant drop in HSP72 expression 2, 4, and 6 hrs after heat stress plus HEMA exposure. HSP72 expression decreased in a dose-dependent fashion, falling at least 50% at 6 hrs after 10 mmol/L exposure to HEMA. The results for TEGDMA were similar to those for HEMA (Figs. 3 and 4), except that the suppression of the HSP72 expression by TEGDMA was more potent, suppressing the six-hour expression over 80% compared with controls not treated with TEGDMA. The TEGDMA effects were also dose-dependent, but occurred at doses one-tenth those of HEMA. Similar trends in HSP72 suppression were seen with HEMA and TEGDMA for the other recovery times (each relative to its corresponding control), although the trends were not always perfect.
The highest concentrations of monomers tested in the present study approached cytotoxic concentrations when SDH activity was used as a measure of cellular activity and at 24-hour exposure times (Yoshii, 1997). With an exposure time of not more than 7 hrs, the resin components were not highly cytotoxic, as measured by SDH activity (Fig. 1  ). Although a synergism between the stress of the material and heat might have been expected to decrease SDH activity more than with the components alone, this result was not observed. In most cases, the combination of heat and resin was similar to heat or resin alone. The exceptions to this trend were HEMA and TEGDMA at their highest concentrations (Fig. 1). The lack of effect of heat on SDH activity indicates that these stress conditions do not alter the energy-producing capability of these cells. Based on the assumption that mitochondrial activity is central to the survival of the cell, the conditions of heat and chemicals applied in the current study appeared to be below lethal levels.
Exposure to chemicals and heat tends to cause errors in protein production or production of immature proteins in cells (Fink, 1999; Yenari et al., 1999). HSP72 has an essential role in assisting protein folding and refolding and is thought to aid in preventing or repairing protein errors under conditions of cellular stress (Polla et al., 1995). HSPs are induced by activation of heat shock factor in the cytoplasm, which is then transferred to the nucleus, where it binds heat shock elements, leading to transcription and synthesis of HSP72 and other HSPs (Yenari et al., 1999). In the current THP-1 model, heat stress of 43°C for 1 hr induced HSP72 fully at 4 and 6 hrs, with minimal expression at 2 hrs (Figs. 2, 3
HEMA and TEGDMA significantly suppressed HSP72 even at apparently sublethal concentrations (Fig. 3 The current study does not address the mechanism by which HEMA and TEGDMA modulate HSP72 expression. Recent results show that HEMA and TEGDMA damage the nucleus (Schweikl et al., 2001) and therefore may alter the activity of the heat shock element and the levels of HSP. Alternatively, HEMA and TEGDMA could inhibit phosphorylation of the heat shock factor, thereby limiting levels of HSP. The role of these or other mechanisms for HSP72 inhibition cannot be determined from the current data. Furthermore, it is currently not known if HEMA and TEGDMA modulate the expression of other HSPs. HEMA and TEGDMA modulated heat-induced HSP72 at concentrations from 2.5 to 10 mmol/L for HEMA and 0.25 to 1 mmol/L for TEGDMA. Although these concentrations appear higher than would be observed clinically, closer analysis supports their relevance to the clinical situation. Dentinal adhesive primers are mostly HEMA, and pure HEMA is approximately 8 mol/L. If used in a direct pulp-capping procedure, then this concentration is clearly higher than those used in the current study. However, the primer is more often used on intact dentin. HEMA has been shown to have a dilution factor of 1000-5000 times across 0.5 mm of dentin with a 10-cm H2O back-pressure (Bouillaguet et al., 1996). Thus, the concentration of HEMA diffusing across the dentin would be on the order of 1.5-8 mmol/L. These concentrations are similar to those used in the current study. TEGDMA is a component of many dentinal bonding resins in approximately 50% concentration. Since pure TEGDMA has a 3.8 mol/L concentration, many bonding resins would have approximately a 2 mol/L concentration of TEGDMA. The dilution of TEGDMA across 0.5 mm of dentin has been shown to be approximately 500 times (Hanks et al., 1994). Thus, the concentration of TEGDMA reaching the pulp would be on the order of 4 mmol/L, again within the range used in the current study. Although there are many factors to consider when converting the in vitro results of the current study to an in vivo situation, the concentrations of monomers used in the current study are plausible. In summary, the current study has shown that several components released from dentinal adhesives can significantly modulate the expression of HSP72 at sublethal concentrations in monocytes. The response can be enhanced or suppressed, depending on the component, its concentration, and time after heat stress.
The authors thank the Ministry of Science and Education of Japan (Grant #13877349) and the Medical College of Georgia Biocompatibility Program and Metalor Corporation for their financial support. The authors also thank Dr. David Pashley for his critical review of the manuscript. Received for publication June 20, 2001. Revision received February 11, 2002. Accepted for publication February 13, 2002.
Journal of Dental Research, Vol. 81, No. 4,
265-269 (2002)
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ABSTRACT
INTRODUCTION
= 0.05) to compare SDH activities at different concentrations. We repeated the entire experiment once to ensure reproducibility. 
