Wyatt R. Hume

Resin Monomer 2-Hydroxyethyl Methacrylate (HEMA) is a Potent Inducer of Apoptotic Cell Death in Human and Mouse Cells

Mechanisms by which the resin monomer 2-hydroxyethyl methacrylate (HEMA) induces hypersensitivity reactions in humans are not well-established, nor have the direct effects of HEMA on cell death been fully characterized. The objective of this study was to establish whether HEMA is capable of inducing apoptotic cell death, and whether differences exist in the levels of apoptotic death induced by HEMA in cells obtained from healthy individuals and from patients with established HEMA hypersensitivity. HEMA induced apoptotic death in Peripheral Blood Mononuclear Cells (PBMCs) obtained from both healthy and HEMA-sensitized patients and in the murine RAW cells in a dose-dependent manner. However, induction of cell death by HEMA was lower in PBMCs obtained from patients in comparison with healthy individuals. Studies reported in this paper demonstrate that HEMA induces apoptotic death, and that decreased susceptibility of lymphocytes to HEMA-mediated death might be an important mechanism for the generation and persistence of hypersensitivity reactions in patients.

Induction of Apoptotic Cell Death in Peripheral Blood Mononuclear and Polymorphonuclear Cells by an Oral Bacterium,Fusobacterium nucleatum

ABSTRACT It is largely unknown why a variety of bacteria present in the oral cavity are capable of establishing themselves in the periodontal pockets of nonimmunocompromised individuals in the presence of competent immune effector cells. In this paper we present evidence for the immunosuppressive role of Fusobacterium nucleatum, a gram-negative oral bacterium which plays an important role in the generation of periodontal disease. Our studies indicate that the immunosuppressive role of F. nucleatum is largely due to the ability of this organism to induce apoptotic cell death in peripheral blood mononuclear cells (PBMCs) and in polymorphonuclear cells (PMNs). F. nucleatum treatment induced apoptosis of PBMCs and PMNs as assessed by an increase in subdiploid DNA content determined by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assays. The ability of F. nucleatum to induce apoptosis was abolished by either heat treatment or proteinase digestion but was retained after formaldehyde treatment, suggesting that a heat-labile surface protein component is responsible for bacterium-mediated cell apoptosis. The data also indicated that F. nucleatum-induced cell apoptosis requires activation of caspases and is protected by NF-κB. Possible mechanisms of F. nucleatums role in the pathogenesis of periodontal disease are discussed.

Cytotoxicity of dental composite components and mercury compounds in lung cells

OBJECTIVE The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA), as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) was investigated on the release of lactatedehydrogenase (LDH) from alveolar epithelial lung cell lines in vitro. METHODS The confluent cell layers from the A549 (human, malignant) and the L2 cells (rat) were incubated with various concentrations of HEMA, TEGDMA, MeHgCl and HgCl2 at 37 degrees C in 2% (v/v) CO2 atmosphere for 8h. In further experiments the L2 cells were incubated with the same compounds for 6-48 h. LDH release was measured and the values were expressed as percentage of the LDH content. The values were plotted on a concentration log-scale and the substance concentration at the maximum slope was assessed as effective concentration (EC50). RESULTS A significant (p<0.05) increase in the LDH release was found in the L2 cells after 8-h incubation with HEMA (4 mmol/l), TEGDMA (2 mmol/l), MeHgCl (0.01 mmol/l) and HgCl2 (0.015 mmol/l), and in A549 cells with HEMA (14 mmol/l), TEGDMA (15 mmol/l), MeHgCl (0.15 mmol/l) and HgCl2 (0.05 mmol/l), compared to controls. The EC50 values from compounds in the L2 cells are shown in the following table (mean; sem in parentheses; n=3-6; #n=1): [see text]. SIGNIFICANCE The toxic effect of HgCl2 and MeHgCl from the L2 cells was about 100-700-fold higher than of the dental composite components. A significant (p<0.05) time dependent increase of toxicity was observed with TEGDMA, HEMA and MeHgCl.

Effect of Eugenol on Constrictor Responses in Blood Vessels of the Rabbit Ear

Aqueous solutions of eugenol depressed vasoconstrictor responses to exogenous norepinephrine (NE), histamine, and peri-arterial sympathetic nerve stimulation in the isolated central artery of the rabbit ear and in the isolated perfused whole rabbit ear. The depression was dose- and time-dependent, and was slowly reversible. Eugenol appeared to act as an inhibitor of smooth muscle contractility.

Distribution and Excretion of TEGDMA in Guinea Pigs and Mice

The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14 CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis.

Uptake, clearance and metabolism of TEGDMA in guinea pigs

OBJECTIVE The resin monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based bonding, cementing and direct tooth filling materials. METHODS In the present study the uptake and the clearance of 14C-TEGDMA applied via different routes were examined in vivo in guinea pigs. TEGDMA (0.02 mmol/kg by weight labeled with a tracer dose 14C-TEGDMA 0.7Bq/g by weight) was administered by gastric tube or by subcutaneous injection. Urine, feces, and exhaled carbon dioxide were collected for 24h after administration. The animals were killed 24h after the beginning of the experiment and various organs removed and 14C-radioactivity measured. RESULTS It was apparent that 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration and was widely distributed in the body following administration by each of the routes. Clearance from most tissues following gastric and intradermal administration was essentially complete within one day. Low fecal 14C-levels (<1% of the administered dose) and urinary levels of about 15% after 24h were noted with each route of administration. Direct measurement of exhaled carbon dioxide showed that 60-65% of the administered dose of 14C left the body via the lungs during 24h. It is likely that 14C-pyruvate is formed in vivo resulting possibly in the formation of toxic 14C-TEGDMA-intermediates. SIGNIFICANCE Despite using a high administered dose, the peak TEGDMA levels in all tissues examined after 24h were at least 100,000-fold less than known toxic levels.

Analyses of Streptococcus mutans in Saliva with Species-Specific Monoclonal Antibodies

Three species-specific monoclonal antibodies (MAbs) against Streptococcus mutans were used to detect and quantify S. mutans levels in saliva. This study shows that MAb-based salivary S. mutans tests exhibit significantly higher specificity and sensitivity than the commonly used selective culture method. Examination of nearly 2,000 human saliva samples shows that S. mutans counts in human saliva vary from less than 10,000 to a high 36 million cells/mL. Over 15% of the saliva samples examined have salivary S. mutans counts over 500,000 cells/mL. When saliva samples were collected at different time points during a day, the number of salivary S. mutans in the same human subject varied, especially before and after sugar uptake. Additionally, data obtained from stimulated versus unstimulated saliva in the same human subjects differed greatly and appear to be completely uncorrelated. This study provides useful information and tools for analyzing the role of S. mutans in human dental caries.

Toxicokinetic of HEMA in guinea pigs.

OBJECTIVE Unconverted 2-hydroxyethylmethacrylate (HEMA) can be released from dental resin materials and can enter the body in humans. In the present study the uptake, distribution and excretion of 14C-HEMA applied via different routes were examined in vivo in guinea pigs. METHODS HEMA (0.02 mmol/kg bw labelled with a tracer dose 14C-HEMA 0.3 Bq/g bw) was administered by gastric tube or by subcutaneous injection. Urine, feces, and exhaled carbon dioxide were collected for 24 h after administration. Guinea pigs were killed 24 h after the beginning of the experiment and various organs removed and 14C radioactivity measured. RESULTS Low fecal 14C levels (about 2% of the dose) and urinary levels of about 15% after 24 h were noted with either route of administration. Direct measurement of exhaled CO(2) showed that about 70% of the dose left the body via the lungs. Two pathways for the metabolism of 14C-HEMA can be described. It is likely that 14C-pyruvate is formed in vivo resulting in the formation of toxic 14C-HEMA intermediates. 14C-HEMA was taken up rapidly from the stomach and small intestine after gastric administration and was widely distributed in the body following administration by each of the routes. CONCLUSIONS Clearance from most tissues following gastric and intradermal administration was essentially complete within one day. The peak HEMA levels in all tissues examined after 24 h were at least onemillion-fold less than known toxic levels.

N-Acetyl Cysteine Mediates Protection from 2-Hydroxyethyl Methacrylate Induced Apoptosis via Nuclear Factor Kappa B-Dependent and Independent Pathways: Potential Involvement of JNK

The mechanisms by which resin based materials induce adverse effects in patients have not been completely elucidated. Here we show that 2-hydroxyethyl methacrylate (HEMA) induces apoptotic cell death in oral keratinocytes. Functional loss and cell death induced by HEMA was significantly inhibited in the presence of N-acetyl cysteine (NAC) treatment. NAC also prevented HEMA mediated decrease in vascular endothelial growth factor secretion. The protective effect of NAC was partly related to its ability to induce NF-kappaB in the cells, since HEMA mediated inhibition of nuclear NF-kappaB expression and function was significantly blocked in the presence of NAC treatment. Moreover, blocking of nuclear translocation of NF-kappaB in oral keratinocytes sensitized these cells to HEMA mediated apoptosis. In addition, since NAC was capable of rescuing close to 50% of NF-kappaB knockdown cells from HEMA mediated cell death, there is, therefore, an NF-kappaB independent pathway of protection from HEMA mediated cell death by NAC. NAC mediated prevention of HEMA induced cell death in NF-kappaB knockdown cells was correlated with a decreased induction of c-Jun N-terminal kinase (JNK) activity since NAC inhibited HEMA mediated increase in JNK levels. Furthermore, the addition of a pharmacologic JNK inhibitor to HEMA treated cells prevented cell death and restored NF-kappaB knockdown cell function significantly. Therefore, NAC protects oral keratinocytes from the toxic effects of HEMA through NF-kappaB dependent and independent pathways. Moreover, our data suggest the potential involvement of JNK pathway in NAC mediated protection.

Mechanisms of N-acetyl Cysteine–mediated Protection From 2-Hydroxyethyl Methacrylate–induced Apoptosis

Resin-based materials are now commonly used in dentistry in restorative materials as well as in endodontic sealers. These materials have been shown to be cytotoxic. The mechanisms by which resin-based materials mediate their adverse effects have not been completely elucidated. Here we show that 2-hydroxyethyl methacrylate (HEMA) induces apoptotic cell death in oral keratinocytes and immune cells through the intrinsic cell death pathway. Functional loss and cell death induced by HEMA was significantly inhibited in the presence of N-acetyl cysteine (NAC) treatment. In addition, HEMA induced a decrease in mitochondrial membrane potential, and an increase in cleaved caspases was potently inhibited in the presence of NAC treatment. Overall, the results reported in this article indicate that NAC is an effective chemoprotectant that can safely be used to protect the pulp and the surrounding tissues from adverse effects of dental restorative and endodontic materials.