HEMA-induced cytotoxicity: oxidative stress, genotoxicity and apoptosis

M. Gallorini, A. Cataldi & V. di Giacomo Department of Pharmacy, University “G. d’Annunzio”, Chieti, Italy

Abstract Gallorini M, Cataldi A, di Giacomo V. HEMA-induced cytotoxicity: oxidative stress, genotoxicity and apoptosis. International Endodontic Journal, 47, 813–818, 2014.

Dental resin composites consist of organic polymers with inorganic fillers used as bonding resins and direct filling materials in dentine adhesives and as sealing agents for inlays, crowns and orthodontic brackets. Despite various modifications in the formulation, the chemical composition of composite resins includes inorganic filler particles and additives, which are incorporated into a mixture of an organic resin matrix. Among them, 2-hydroxyethylmethacrylate (HEMA) is one of the most frequently used. Several studies have attempted to clarify the mechanisms underlying HEMA cytotoxicity. Most of them support the hypothesis that this compound, once released in the oral environment, increases reactive oxygen species (ROS) production and

Introduction Increasing numbers of resin-based dental restorative materials have been developed over the past decade. It has been found that each resin-based material releases several components into the oral environment. In particular, the hydrophilic monomer, 2-hydroxyethylmethacrylate (HEMA), is leached out from various composite resins and adhesive materials in considerable amounts during the first 24 h after

oxidative DNA damage through double-strand breaks evidenced by in vitro presence of micronuclei. As a consequence, the glutathione detoxifying intracellular pool forms adducts with HEMA through its cysteine motif and inflammation begins to occur: transcription of early genes of inflammation such as tumour necrosis factor a or inducible cyclooxygenase up to the secretion of prostaglandins 2. These phenomena are counteracted by N-acetylcysteine (NAC), a nonenzymatic antioxidant, but not by vitamin E or other antioxidant. Consequently, NAC prevents HEMA-induced apoptosis acting as a direct ROS scavenger. This minireview collects the most significant papers on HEMA and tries to make an overview of its cytotoxicity on different cell types and experimental models. Keywords: apoptosis, genotoxicity, HEMA, oxidative stress, ROS. Received 30 October 2013; accepted 16 December 2013

polymerization (Geurtsen 2000). Moreover, HEMA, together with tetraethylenglycol dimethacrylate (TEGDMA), is able to diffuse through dentine into the pulp space at significantly high concentrations in the millimolar range (Bakopoulou et al. 2009). In vitro studies revealed genotoxic, mutagenic, oestrogenic and teratogenic effects of these composites components (Krifka et al. 2013). This minireview aims to investigate the main mechanisms that underlie HEMA cytotoxicity in different in vitro experimental models.

HEMA-induced oxidative stress Correspondence: Dr Viviana di Giacomo, Department of Pharmacy, University “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy (Tel.: +3908713554509; e-mail: [email protected]).

© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

In several in vitro studies, the toxicity of HEMA has been investigated, and its binding to glutathione (GSH) cysteine has been identified as a key event in the toxic

International Endodontic Journal, 47, 813–818, 2014


HEMA and cytotoxicity Gallorini et al.

response (Ansteinsson et al. 2013) (Fig. 1). Glutathione is a tripeptide with a c peptide linkage between the amino group of cysteine and the carboxyl group of the glutamate side chain. It is an antioxidant, preventing damage to important cellular components caused by reactive oxygen species (ROS), such as free radicals and peroxides (Circu & Aw 2012). GSH can also detoxify xenobiotics, such as methacrylates, by conjugating, via a Michael addition, the thiol group with their a-b-unsaturated carbon–carbon moiety. The reaction is catalysed by glutathione S-transferase, present in cytosol, microsomes and mitochondria, that is involved in detoxification processes (Toroser et al. 2006). This reaction has been observed in several in vitro studies employing different kind of cell cultures (Engelmann et al. 2002, Nocca et al. 2007) (Table 1). HEMA at a concentration of 1.1 mmol L 1 does not affect HL-60 proliferation and viability, but it acts as a cellular differentiating agent only in the absence of N-acetylcysteine (NAC) (Nocca et al. 2007). HEMA causes a decrease in the oxygen consumption rate in intact cells, completely inhibited by NAC, and in isolated mitochondria. Nocca et al. (2011) presented a method to detect and identify GSH-methacrylate adducts obtained via a Michael addition reaction. They found that the formation of adducts was associated with different kinetics, when the reaction occurs in human gingival fibroblasts (HGFs) or in erythrocytes, compared with methacrylates mixture with glutathione without cells, suggesting that the reaction is strongly correlated with glutathione S-transferase. Previous studies hypothesized that low concentrations of various resin (co)monomers

Figure 1 HEMA-induced oxidative stress through GSH covalent binding. GST: glutathione S-transferase. GSSG: glutathione disulphide, derived from two glutathione molecules. GPx: glutathione peroxidase. SOD: superoxide dismutase.


International Endodontic Journal, 47, 813–818, 2014

accumulate intracellularly within a short period of time to an overall toxic level, which then results in an irreversibile exhaustion of the cells detoxifying GSH pool, thus finally causing apoptosis (Lander et al. 1997, Miyamoto et al. 2003). Moreover, HEMA triggers the expression of stress-responsive genes at the transcriptional level through the accumulation of ROS. Krifka et al. (2012) concluded that monomer-induced apoptosis is an active cell response to levels of ROS exceeding the cells’ ability to maintain redox homeostasis. They hypothesized that cells differentially activate a balance network of enzymatic cellular antioxidants to control the intracellular oxidative state after exposure to HEMA monomer. It has also been shown that HEMA increases the production of reactive oxygen species, which can cause inflammation (Table 2) and a delay in cell cycle progression (Chang et al. 2005, Di Nisio et al. 2013). Spagnuolo et al. (2004) showed that HEMA-induced ROS production is very important to modulate the activation of nuclear factor kappa B (NF-kB), which plays a protective role to counteract HEMA cytotoxicity.

HEMA-induced DNA damage In another study, that tested HEMA effects on V79-4 Chinese hamster lung fibroblasts and RPC-C2A pulp cells, ROS production was correlated with DNA fragmentation and to a dose-related induction of micronuclei, confirming that HEMA-induced apoptosis, as well as mutagenicity of HEMA, may be mediated by oxidative stress (Lee et al. 2009). The molecular mechanism by which resin monomers induce genotoxicity has not been completely elucidated. DNA-reactive acrylate may cause partial deletions in the genome of mammalian cells by direct binding to DNA (Dearfield et al. 1991). To test the hypothesis that HEMA-induced ROS production is correlated with its genotoxicity, the frequencies of micronucleated cells induced by the resin monomers were observed in the absence and presence of N-acetylcysteine (Lee et al. 2009). The significant decrease in the number of micronucleated cells, when co-treated with NAC, supports the role of ROS in mutagenicity of the resin monomers. In addition to detrimental effects on lipids and proteins, ROS likely interact with parts of DNA, such as sugar moieties, chromatin proteins, as well as pyrimidines and purines, which may be rapidly fixed by base or nucleotide excision repair. More severe damage, in turn, causes single- or doublestrand breaks, base modifications and DNA protein crosslinks, which consequently impair the stability of

© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

Gallorini et al. HEMA and cytotoxicity

Table 1 HEMA cytotoxicity through GSH adduct formation HEMA concentration 1

Experimental model


Up to 15 mmol L for 24 h (Walther et al. 2004) 0–16 mmol L 1 for 24 h (Ansteinsson et al. 2013) 1 mmol L 1 for 48 h (Nocca et al. 2011) 0.1–10 mmol L 1 for 4 h (Volk et al. 2006) 1.10 mmol L 1 for 24 and 48 h (Nocca et al. 2007) 6 and 8 mmol L 1 for 4 h (Krifka et al. 2012)

11Lu cells, 16Lu cells, L2 cells, A549 cells BEAS 2B cells

GSH depletion

HGFs, erithrocytes and synthetic GSH-methacrylates adducts HGFs

GSH-methacrylates adduct formation


0–8 mmol L 1 for 30 min (Samuelsen et al. 2010)

SM 10–12

Cellular differentiation and decrease in oxygen consumption, GSH depletion stimulating G6PDH and GR Inhibition of GPx1/2 expression further reduced in the presence of BSO, inhibition of SOD1 expression and upregulation of catalase expression Increased level of ROS and of GSH-HEMA complex formation

GSH-methacrylates adduct formation, GSH depletion

GSH depletion at a concentration of 1.6 mmol L

RAW264.7 mouse macrophages


11Lu and 16Lu cells, human fibroblast-like lung cells; L2 cells, rat epithelial-like lung cells; A549 cells, adenocarcinomic human alveolar basal epithelial cells; BEAS 2B cells, human epithelial-like lung cells; HGFs, primary human gingival fibroblasts; HL-60, human promyelocytic leukaemia cells; G6PDH, glucose-6-phosphate dehydrogenase; GR, glutathione reductase; RAW264.7, mouse leukaemic monocytes macrophage cell line; GPx 1/2, glutathione peroxidase 1/2; BSO, buthionine sulphoximine; SOD1, superoxide dismutase; SM 10–12, murine red blood cell line.

Table 2 HEMA-induced oxidative stress and inflammation HEMA concentration 1

3 mmol L for 24 and 96 h (Di Nisio et al. 2013) 0–10 mmol L 1 for 24 h (Dong et al. 2006) 3 mmol L 1 for 24 and 96 h (Cataldi et al. 2013) 0–12 mmol L 1 for 24 h (Lee et al. 2009)

Experimental model HGFs V794 fibroblasts and RPC-C2A pulp cells HGFs RAW 264.7 cells

Effects Increase in ROS production, TNF a and COX-2 gene expression and PGE2 release. Dose- and oxidative stress-dependent genotoxicity counteracted by NAC. Cell proliferation decrease, increase in ROS production, PKC a activation, and iNOS and Bax expression. Dose-dependent increase in COX-2 gene expression after 5 h of exposure, upregulation of COX-2 proteins.

HGFs, primary human gingival fibroblasts; ROS, reactive oxygen species; TNF a, tumour necrosis factor alpha; PGE2, prostaglandin 2; V794 fibroblasts, Chinese hamster fibroblast-like lung cells; RPC-C2A pulp cells, rat clonal dental pulp cells; NAC, N-acetylcysteine; PKC a, protein kinase C alpha isoform; iNOS, inducible nitric oxide synthase; Bisindolylmaleimide VIII, PKC a pharmacological inhibitor; RAW264.7, mouse leukaemic monocytes macrophage cell line; COX-2, inducible cyclooxygenase.

the genome (Krifka et al. 2013). Resin monomers such as HEMA are responsible for the formation of DNA double-strand breaks. This genome damage leads to the activation of ataxia telangiectasia mutated (ATM) gene, a member of the phosphoinositide 3-kinase-like family of serine/threonine protein kinases, by autophosphorylation after long exposition periods to resin monomers (Eckhardt et al. 2009). Ansteinsson et al. (2011) demonstrated that in response to HEMA-induced DNA damage, a number of cell cycle checkpoints are activated leading to complex kinase-signalling networks that reduce the progression through the cell cycle and, in parallel, also mediate recruitment of DNA repair pathways. Activated ATM usually targets many cell cycle

© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

checkpoints such as the checkpoint kinase 2 (Chk2) or the histone H2AX that results in a p53-dependent apoptosis. They found that HEMA inhibits the proliferation of human bronchial epithelial BEAS 2B cells as a result of an S-phase accumulation caused by DNA damage that was followed by phosphorylation of H2AX, Chk2 and p53. The S-phase accumulation is not counteracted by Trolox (vitamin E), indicating that an increased ROS level is not directly involved. In another study, HEMA induced significant DNA migration, detected by the comet assay, in human salivary glands as human target cells of carcinogenesis (Kleinsasser et al. 2006), as previously reported in lymphocytes (Kleinsasser et al. 2004). Both HEMA and methacrylic acid (MAA), HEMA hydrolysis

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product, evoke DNA damage in HGFs, resulting in a significant fragmentation of DNA in the alkaline version of the comet assay, confirming an MAA-dependent genotoxicity of composite resin monomers (Szczepanska et al. 2012). Unlike TEGDMA, HEMAinduced arrest of cell cycle in G2 phase is counteracted by antioxidants. Schweikl et al. (2007) demonstrated a reversal in cell cycle delay in HEMA-treated cell culture in the presence of 10 Mmol L 1 N-acetylcysteine. An in vivo study investigated the genotoxicity of four resin composite materials, including HEMA (Arossi et al. 2010). No induction of gene mutations was detected with HEMA, although high concentrations of HEMA induced micronuclei formation in vitro.

HEMA-induced apoptosis in vitro It has been largely reported that HEMA induces apoptosis in vitro after 24 h (Table 3) (Hanks et al. 1991, Krifka et al. 2012). Moreover, HEMA induces morphological changes in cultured cells, such as human pulp fibroblasts (HPFs) and human gingival epithelial cells. HPFs treated with 10 mmol L 1 HEMA presented apparent retraction and loss of filopodia and lamellopodia, the extended cellular processes, which are crucial for cell proliferation and movement during wound healing as well as tissue morphogenesis (Chang et al. 2005). HEMA treatment at relatively high concentrations, for example, 5-10 mmol L 1, results in ROS formation and concentration-dependent apoptosis as well

as phosphorylation of the mitogen-activated protein kinases extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, that is reversed by their selective inhibitors (Samuelsen et al. 2007). The stress-activated protein kinases (SAPKs) JNK and p38 have been related mainly to oxidative stress and apoptosis, as well as to inflammatory responses following exposure to chemical agents, while it has been reported a role of ERK in apoptosis. The protective effect of N-acetylcysteine is partly related to its ability to induce NF-kB translocation. Moreover, blocking of NF-kB nuclear translocation in oral keratinocytes sensitizes these cells to HEMA-mediated apoptosis (Paranjpe et al. 2009). NAC-mediated prevention of HEMA-induced cell death in NF-kB knockdown cells is correlated with a decreased induction of JNK (Paranjpe et al. 2009). Moreover, N-acetylcysteine inhibits HEMA-mediated toxicity through induction of differentiation in dental pulp stromal cells (DPSCs) as the genes for dentine sialoprotein, osteopontin (OPN), osteocalcin, and alkaline phosphatase, which are induced during differentiation, are also induced by NAC (Paranjpe et al. 2007). More importantly, when added either alone or in combination with HEMA, vitamin E and vitamin C do not increase the gene expression of OPN; in addition vitamin E inhibits the protective effect of NAC on DPSCs, and this compound fails to prevent either HEMA mediated cell death or decrease in vascular endothelial growth factor secretion by human DPSCs (Paranjpe et al. 2007).

Table 3 HEMA-induced apoptosis and differentiation HEMA concentration 1

3.8–15 mmol L from 2 to 16 h (Samuelsen et al. 2007) 8.2 mmol L 1 from 4 to 24 h (Paranjpe et al. 2009) 0–10 mmol L 1 for 24 h (Kim et al. 2011) 10 mmol L 1 from 30 min to 24 h (Spagnuolo et al. 2008) 3 mmol L 1 for 48 and 72 h (Zara et al. 2011) 16.4 mmol L 1 for 0–18 h (Paranjpe et al. 2008)

Experimental model SM 10–12 cells 293T and HEp2 cells MG63 cells HPCs HGFs cocultured with S. mitis Oral keratinocytes and immune cells

Effects ROS formation and concentration-dependent apoptosis as well as ERK, p38 and JNK activation. Functional loss and apoptosis inhibited by NAC, decrease in VEGF secretion, inhibition of NF-kB expression and translocation. Reduced ALP activity and OPN mRNA expression, downregulation of GSH, inhibition of Nrf2 nuclear translocation. Dose-dependent increase in apoptosis, increased ERK 1/2 phosphorylation, decreased AKT phosphorylation. Apoptosis induction and pro-collagen production decrease counteracted by bacteria. Decrease in mitochondrial membrane potential and increase in cleaved caspases potently inhibited in the presence of NAC treatment.

SM 10–12 cells, murine red blood cell line; ROS, reactive oxygen species; ERK 1/2, extracellular signal-regulated kinase 1/2; p38, mitogen-activated protein kinase, cytokine-specific binding protein; JNK, c-Jun N-terminal kinase; 293T cells, human embryonic kidney cell line; HEp2 cells, human epidermoid cancer cells; VEGF, vascular endothelial growth factor; NF-kB, nuclear factor kappa-light-chain enhancer of activated B cells; ALP, alkaline phosphatase; OPN, osteopontin; MG63 cells, human osteosarcoma cell line; Nrf2, nuclear factor (erithroid-derived 2)-like 2; HPCs, human primary pulp fibroblasts; AKT, protein kinase B; HGFs, primary human gingival fibroblasts.


International Endodontic Journal, 47, 813–818, 2014

© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

Gallorini et al. HEMA and cytotoxicity

HEMA-induced cytotoxicity in HGFs/ S.mitis coculture model Even if HEMA cytotoxicity is well known and its mechanisms have been largely elucidated in vitro, recent studies demonstrated a decrease in HEMA effects when human gingival fibroblasts are cocultured with Streptococcus mitis (Table 2), a physiological commensal of human oropharynx (Zara et al. 2011, Di Giulio et al. 2012). The cocultivation of S. mitis strains/HGFs produces a decrease in the rate of HGFs death, both at 48 and 72 h assessed by trypan blue dye. Moreover, the addition of saliva to the coculture system improves the protective role of bacteria on eukaryotic culture against HEMA cytotoxicity (Di Giulio et al. 2013). HEMA treatment increases the adhesion between S. mitis and human gingival fibroblasts, which seems to be mediated by the PKC a/integrin b 1 signalling system, improved by the presence of saliva. It also reduces the viability and the adhesion of HGFs to polypropylene substrate in terms of procollagen I and matrix metallopeptidase 3 expression. The presence of saliva and S. mitis reduces the number of necrotic fibroblasts and upregulates the expression of both procollagen I and matrix metallopeptidase 3 (di Giacomo et al. 2013).

Conclusions Studies on the molecular toxicology of substances released by resin-based dental restorative materials clearly support that they are able to cause cytotoxic and genotoxic effects at concentrations relevant to those released into the oral cavity (Bakopoulou et al. 2009). Further understanding of the cellular mechanisms involved in these processes as well as the clarification of the causal relationship between the availability of glutathione and monomer-induced apoptosis will stimulate a constructive debate on the development of smart dental restorative materials (Krifka et al. 2013). Moreover, the clinical significance of in vitro mutagenity and genotoxicity data and the possibility for systemic mutagenic effects should be further investigated in animal models.

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© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

HEMA-induced cytotoxicity: oxidative stress, genotoxicity and apoptosis.

Dental resin composites consist of organic polymers with inorganic fillers used as bonding resins and direct filling materials in dentine adhesives an...
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