Human lymphocytes were exposed to HEMA and TEGDMA for 24 h.
Cytotoxic effects were measured only in TEGDMA (100 μM) treated cells.
Genotoxic effects were detected in the comet assay at the highest concentrations.
No increase in the micronucleus frequency or an arrest of the cell cycle was shown.
Chromosome aberrations and SCEs were recorded at lowest concentrations tested.
Residual unbound resin monomers of 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) are known to diffuse in the saliva and through dentin and pulp into the blood and may affect cellular integrity. The current study was performed to investigate the genotoxic potential of both monomers in distinctly lower concentrations than known to cause cytotoxic damage.
Lymphocytes from 10 healthy volunteers were treated with HEMA (10 μM–1 mM) and TEGDMA (1 μM–100 μM) for 24 h. Cell viability, apoptosis and influence on cell cycle kinetics were assessed by flowcytometry. DNA damage was determined by the alkali version of the comet assay in combination with the FPG protein and by the cytokinesis-block micronucleus (CBMN) test. Additionally, the chromosome aberration (CA) test and sister chromatid exchange (SCE) test were performed.
A slight decrease in cell viability was detected only at the highest concentration of TEGDMA. Genotoxic effects were measurable in the comet assay at 1 mM of HEMA and 100 μM of TEGDMA, with and without FPG protein, but not in the CBMN test or the cell cycle analysis. Contrary to these findings, a significant dose-dependent increase in the frequency of CAs and SCEs could be demonstrated in all tested concentrations.
This is the first time clastogenic responses to HEMA and TEGDMA have been detected in concentrations distinctly lower than those reported for causing cytotoxic or even genotoxic effects. These findings underline the importance of using test batteries with different genotoxicological endpoints to describe the multiple effects of both resin monomers.
Dental composite resins are commonly used for the restoration of structural tooth damage due to their esthetic advantages. Inorganic filler particles like quartz, ceramic and silica are incorporated into a resin matrix. This matrix contains viscous monomers, e.g., urethane dimethacrylate (UDMA), and dilutive monomers like 2-hydroxyethyl methacrylate (HEMA) and the comonomer triethylene glycol dimethacrylate (TEGDMA) . Their original contents vary between 25% and 55%. During and after the polymerization process, residual resin monomers and other components remain unbound and are released from dental composite materials into the oral environment. After minutes and hours, HEMA and TEGDMA are detectable in microgram quantities in the saliva . After longer periods, hydrolytic disintegration and unspecific salivary esterases may contribute to a further release of resin monomers, which are able to diffuse through dentin and pulp in concentrations effecting cellular functions . However, systemic blood concentrations are below detection level . Corresponding to dentin thickness and grade of polymerization, concentration of HEMA in the pulp may reach levels between 1.5 and 8 mM and TEGDMA in the range of 4 mM .
These findings have encouraged investigations regarding the potential cytotoxicity and genotoxicity of the major monomers, co-monomers and composites: cytotoxic and genotoxic effects in the organic matrix of composite materials were reviewed in . Among others, cytotoxic effects like inhibition of DNA and protein synthesis for TEGDMA were shown in mammalian fibroblasts . A direct reduction in cell viability in HeLa S3 cells using the MTT assay was reported for HEMA . Further investigations have also supported these findings in human lymphocytes, salivary gland cells and different cell lines . Although no severe systemic adverse effects of monomers are described, some monomers were observed to participate in allergic reactions like urticaria and allergic contact dermatitis .
A compilation of tests was performed to identify specific compounds that contribute to genotoxicity, e.g., the extracts of the dental materials Vitrebond ® and AH26 ® showed genotoxic responses . A clastogenic potential of HEMA and TEGDMA was demonstrated in V79 Chinese hamster lung fibroblasts after an incubation period of 24 h by a dose-related increase in micronuclei .
In addition to chemical genotoxins, humans are exposed on a daily basis to different physical DNA-damaging mechanisms like ultraviolet radiation, causing DNA lesions such as cross-linking of adjacent pyrimidines, or ionizing radiation, which are known to damage various cell components and inducing a multitude of DNA adducts . Most DNA lesions are rapidly repaired by repair enzymes, e.g., by base or nucleotide excision repair . However, unrepaired DNA damage may be expressed by single- or double-strand breaks, base modifications or DNA cross-links, which could affect the stability of the genome. These mechanisms could result in mutagenesis during DNA replication or cell division, or even in cell cycle arrest followed by apoptosis .
The current study was performed to investigate genotoxic and cytotoxic responses in vitro after treatment with HEMA and TEGDMA in concentrations distinctly lower than previously described to induce cytotoxic effects . Therefore, the chromosome aberration test and the sister chromatid exchange test were included in the test battery for the description of the induced effects in a standard human cell population.