DNA-damage, cell-cycle arrest and apoptosis induced in BEAS-2B cells by 2-hydroxyethyl methacrylate (HEMA)

doi:10.1016/j.mrgentox.2011.04.011

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Volume 723, Issue 2, 16 August 2011, Pages 158-164

https://doi.org/10.1016/j.mrgentox.2011.04.011 Get rights and content

Abstract

The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA) is commonly used in resin-based dental restorative materials. These materials are cured in situ and HEMA and other monomers have been identified in ambient air during dental surgery. In vitro studies have demonstrated a toxic potential of methacrylates, and concerns have been raised regarding possible health effects due to inhalation. In this study we have investigated the mechanisms of HEMA-induced toxicity in the human lung epithelial cell line BEAS-2B. Depletion of cellular glutathione (GSH) and an increased level of reactive oxygen species (ROS) were seen after 2 h of exposure, but the levels were restored to control levels after 12 h. After 24 h, inhibited cell proliferation and apoptotic cell death were found. The results of the Comet assay and the observed phosphorylation of DNA-damage-associated signalling proteins including Chk2, H2AX, and p53 suggest that the toxicity of HEMA is mediated by DNA damage. Further, the antioxidant trolox did not counteract the HEMA-induced cell-cycle arrest, which indicates that the DNA damage is of non-oxidative origin.

Introduction

Resin-based restorative and preventive materials are extensively used in dentistry and the monomer 2-hydroxyethyl methacrylate (HEMA) is common in dental materials designed to polymerize in situ [1]. Incomplete polymerization of resin components, which is inevitable in the clinical situation, can result in release of monomers into surrounding tissues, the saliva and the air [2]. HEMA has also been shown to diffuse rapidly through the dentin into the pulp tissue, thereby causing pulp irritation [3]. Recent studies have elucidated possible adverse health effects upon exposure to monomers via inhalation, suggesting that these compounds may cause asthma and irritation of mucous membranes. Methacrylates, including HEMA, are considered to be possible causative agents [2].

Resin monomers have been identified as cytotoxic compounds in a variety of different in vitro models [3], [4], [5], [6]. Basic cellular functions such as synthesis of protein and DNA as well as enzyme activities have been reported to be altered following exposure to methacrylate monomers [3], [4], [7], [8]. Methacrylates are known to suppress cell proliferation and induce apoptosis [5], [9], [10]. However, the mechanisms involved need to be further explored. We have previously shown that the monomer HEMA caused a delay in cell proliferation and induced apoptosis in the rat salivary gland cells [5]. Resin monomers have also been identified as chemicals that can influence the cellular redox balance by increasing the level of reactive oxygen species (ROS) and depleting the level of glutathione (GSH) [11], [12]. These events have been associated with reduced cell proliferation and increased apoptosis. Furthermore, it has been demonstrated that both HEMA and TEGDMA can cause genotoxic effects. In V79 Chinese hamster lung fibroblasts HEMA was found to induce the formation of micronuclei, but it has failed to induce mutations in bacterial test systems [13].

In response to DNA damage, a number of cell-cycle checkpoints are activated leading to complex kinase-signalling networks that reduce the progression through the cell cycle. In parallel, checkpoint signalling also mediates recruitment of DNA-repair pathways. If the extent of damage exceeds repair capacity, additional signalling cascades are activated to ensure elimination of damaged cells by apoptosis. Ataxia telangiectasia mutated gene (ATM) and ATR (ATM-RAD3-related) are members of the phosphoinositide 3-kinase (PI3-kinase) cell-signalling family. These proteins have an essential role in the detection and signalling of DNA-damage [14]. Activated ATM has a number of downstream targets including the cellular check-point kinase Chk2, as well as histone H2AX, which has been used as a marker of DNA double-strand breaks (DSBs). In contrast, activation of Chk1 is associated with activation of ATR. These phosphorylations may finally activate p53, a key regulator of cell cycling and apoptosis [15].

The aim of this study was to elucidate the mechanisms of HEMA-induced cell-cycle arrest and apoptosis. Our hypothesis is that these events are related to non-oxidative DNA-damage.

Section snippets

Chemicals

LHC9 medium was purchased from Biosource International (Camarillo, CA, USA). Collagen (PureCol™) was purchased from Inamed Biomaterials (Fremont, CA, USA). 2-Hydroxyethyl methacrylate (HEMA) (CAS no.868-77-9), purity ≥ 97% was from Fluka Chemie AG (Buchs, Switzerland). Low melting-point agarose (LMPA) and normal melting-point agarose (NMPA) were purchased from Invitrogen (Carlsbad, CA, USA). The TUNEL Assay (TMR red) was obtained from Roche Diagnostics (Oslo, Norway). Nuclear isolation and

Proliferation, cell-cycle distribution, and apoptosis

The proliferation assay showed that the cell density was reduced to 20% of the control after exposure to 4 mM HEMA (Fig. 1A). Flow-cytometry analysis shows that HEMA (5.4 mM) increased the number of cells in early S-phase of the cell cycle (Fig. 1B and C). Co-treatment of the cells with the antioxidant Trolox (100 μM) did not influence the effect of HEMA (Fig. 1D and E). Based on microscopic examination of cells after a 24-h exposure to HEMA (Fig. 2), the number of apoptotic cells increased with

Discussion

Resin monomers induce biological effects in vitro [5], [6], [10], [21], [22], [23], [24]. However, the underlying mechanism is still not clear. The present study revealed reduced cell density as well as altered cell-cycle distribution and increased cell death after exposure of BEAS-2B cells to HEMA, compared with non-exposed cells. These findings are consistent with previous reports on other cell types [5], [24]. The relative number of apoptotic cells was much lower than the reduction in cell

Conclusion

HEMA inhibited the proliferation of human bronchial epithelial BEAS 2B cells as a result of an S-phase accumulation caused by DNA-damage. The DNA damage was followed by phosphorylation of H2AX, Chk2 and p53. The S-phase accumulation was not counteracted by trolox, indicating that an increased ROS level was not directly involved.

Conflict of interest statement

None.

Acknowledgements

The authors wish to thank Mrs. Inger-Sofie Dragland and Mrs. Else Morisbak (both NIOM) for their excellent laboratory assistance and professor Nils Roar Gjerdet (University of Bergen/NIOM) for valuable discussions.