Effect of dental metal ions on glutathione levels in THP-1 human monocytes.

The release of metal ions from dental restorations has been well established, but the sublethal effects of these ions on oral tissues remain undefined. Metal ions are a potential cause of sublethal oxidative stress in cells. Oxidative stress is known to alter cellular processes which are important in the inflammation of tissues. The presence of oxidative stress is commonly assessed by measuring the oxidized (GSSG) and reduced (GSH) forms of cellular glutathione. Normally, the majority of cellular glutathione exists as GSH and the GSH-GSSG ratio is high. Since monocytes often orchestrate inflammatory responses, oxidative stress in monocytes is important. Our hypothesis for the current work was that metal ions induce oxidative stress in monocytes which is manifested by a lower GSH-GSSG ratio. Human THP-1 monocytes were exposed for 24 h to sublethal concentrations of ions of Ag, Be, Co, Cu, Hg, Ni, Pd, and Zn--all known to be released from dental biomaterials. GSH and GSSG were measured using colorimetric assays. Cu lowered the GSH-GSSG ratio as hypothesized, but the ratio could not be determined for other metals because other metal ions interfered with the GSSG assay. However, a statistically significant (ANOVA/Tukey) increase in GSH per cell was observed upon exposure to Ag (50%), Co (100%), Hg (250%) and Zn (10%). This increase may be indicative of an oxidative stress in its own right. Alterations in GSH levels may be important to how released metal ions alter cytokine secretion from monocytes and other cells which play a role in the inflammatory response to dental biomaterials.

Effect of sub-lethal concentrations of HEMA (2-hydroxyethyl methacrylate) on THP-1 human monocyte-macrophages, in vitro.

OBJECTIVE: Resin monomers such as HEMA (2-hydroxyethyl methacrylate) can be released from restorative materials and diffuse into the tooth pulp over long periods of time. Although the short-term toxicity of resin monomers has been well documented, little is known about the risk for chronic toxicity resulting from low concentrations of resins. Thus, the hypothesis tested in this study was that sub-lethal concentrations of HEMA alter the functions of macrophages after long-term exposure. METHODS: Human THP-1 monocyte-macrophages were exposed to concentrations of HEMA between 0 and 1.5 mmol/l for up to 6 weeks. Cellular proliferation was measured by a hemocytometer with trypan-blue dye exclusion. Mitochondrial activity was measured by the MTT assay, and total cellular protein was measured using the bicinchoninic acid assay. RESULTS: Macrophage proliferation was inhibited 40-50% (significant, p < 0.05) by as little as 0.75 mmol/l after 1 week of exposure. Inhibition of proliferation remained constant after 1 week. The total protein per cell increased by as much as 80% (significant, p < 0.05) after 2 weeks and remained elevated through 6 weeks. Mitochondrial activity per cell increased 60-80% (significant, p < 0.05) after 2 weeks, then decreased. However, mitochondrial activity remained significantly elevated above controls through 6 weeks. SIGNIFICANCE: Findings from the current study indicate that 6-week exposures of monocytes to HEMA alter their proliferation and other activities at concentrations substantially lower than previously reported. This is particularly relevant in light of evidence that such concentrations have been previously shown to come through dentin by diffusion.

Effect of dentin bonding agents on the secretion of inflammatory mediators from macrophages.

Dentin bonding agents (DBAs) have been proposed as substitutes for amalgam as root-end filling materials. The current study tested the hypothesis that certain components of DBAs could alter the secretion of cytokines from macrophages. Such alteration would likely be undesirable for healing of the periapical tissues. Human THP-1 macrophages were exposed to 2-hydroxyethyl methacrylate, 4-methacryloxyethyl trimelliate anhydride, bisphenol-gycidylmethacrylate, and urethane dimethacrylate. The secretion of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) were measured with or without challenge by lipopolysaccharide (LPS). Results showed that all DBA components completely suppressed LPS-induced IL-1 beta and TNF-alpha secretion at concentrations that suppressed mitochondrial activity by 50%. In addition, 4-methacryloxyethyl trimelliate anhydride induced secretion of IL-1 beta, but not TNF-alpha, without the LPS challenge. These results indicate that DBA components may alter normal macrophage-directed inflammatory responses if the macrophages are exposed to sufficiently high concentrations of these components.

Effects of dentin bonding agents on macrophage mitochondrial activity.

Dentin bonding agents (DBA) have been considered for use as root-end fillings. Previous studies have documented the release of DBA components in vivo and in vitro, but the biological implications are not clear. The macrophage is important in wound healing, and likely to be important in any inflammatory response. Therefore, this study determined the concentrations of the components of DBAs that suppress the mitochondrial activity of human macrophages in vitro. THP-1 macrophages were cultured in the presence of four DBA components (2-hydroxyethyl methacrylate (HEMA), 4-methacryloxyethyl trimellitate anhydride (4-META), bisphenol-glycidylmethacrylate (Bis-GMA), and urethane dimethacrylate (UDMA)) at various concentrations and for varying durations. Residual effects were also measured after the resins were removed. Controls received only the vehicle solution, ethanol or water. THP-1 mitochondrial activity was estimated using the MTT assay, and the 50% toxicity concentrations (TC50) were determined graphically. Resin components suppressed the mitochondrial activity of macrophages at different concentrations (TC50 values for HEMA (10,000 mumol/L), 4-META (3,800 mumol/L), Bis-GMA (130 mumol/L), and UDMA (110 mumol/L) at 24 h, and the effect was time-dependent. Residual effects were observed for all resins.