Sister-chromatid exchange, chromosomal aberrations and delays in cell-cycle kinetics in human lymphocytes induced by dental composite resin eluates

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Abstract

We have investigated eluates derived from commercially available composite resin-based materials used for direct (Tetric Ceram/Ivoclar-Vivadent, Simile/Pentron, Filtek Z-250/3M ESPE) and indirect (Adoro/Ivoclar-Vivadent and Conquest Sculpture/Pentron) dental restorations, with respect to their genotoxic effects on human peripheral lymphocytes. Primary lymphocyte cultures obtained from blood samples of three healthy donors were exposed to eluates of freshly cured specimens of all the materials tested. Metaphases were induced with phytohaemagglutinin, collected after a 72-h treatment using colchicine and stained with the Fluorescence Plus Giemsa (FPG) procedure. Preparations were scored for sister-chromatid exchange (SCE) and chromosomal aberrations (CAs). The proliferation rate index (PRI) and the mitotic index (MI) were also calculated.

Our results show that eluates derived from the three direct composites (Filtek Z-250, Simile and Tetric Ceram) increased the frequencies of SCE and CAs and markedly reduced PRI and MI. Tetric Ceram's eluate, being the most genotoxic of all eluates tested, increased the frequencies of SCE up to 24.40 per cell (control, 9.87 per cell) and of CAs up to 424 per 100 metaphases scored (control, 5). Moreover, it caused a pronounced decrease of the PRI down to 1.31 (control, 2.44) and of the MI down to 9.8‰ (control, 19.2‰). In contrast, eluates derived from the laboratory-processed composites (Adoro and Conquest Sculpture) induced much less cytogenetic damage. Overall, the degree of genotoxicity and cytotoxicity decreased as follows: Tetric Ceram > Filtek Z-250 > Simile > Adoro = Conquest Sculpture.

These results indicate that composite resins used for direct and indirect dental restorations differ extensively in their cytotoxic and genotoxic potential and in their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. This underlines the impact of improved polymerization with respect to their biological behavior.

Introduction

Dental composite resin-based materials have been widely studied for cytotoxicity in various cell culture systems [1], [2]. Their cytotoxic effects reported thus far have been attributed to the release of residual monomers or other substances derived either from incomplete polymerization or resin degradation [3], [4]. More than 30 different compounds have been identified as being released from the polymerized dental composites, including major monomers, co-monomers, additives and reaction products, all with different cytotoxic potential [5]. The released components may diffuse through the dentine into the pulp space or, alternatively, they may either come in contact with the gingiva, or be swallowed via the saliva, taken up by the circulating blood and excreted with the urine [6].

There are a few studies dealing with the possible mutagenicity and genotoxicity of various single components released from polymerized composite resins. TEGDMA, bisphenol A and HEMA have been found to increase the number of micronuclei at sub-toxic concentrations and promote degradation of DNA from salivary gland tissue and lymphocytes, as shown by a comet assay [7], [8], [9]. TEGDMA was also reported to induce extensive deletions of sequences in the hypoxanthine-guanine phosphoribosyl-transferase (Hprt) gene in V79 Chinese hamster lung fibroblasts [10]. Other substances released from composite resins, such as the photo-initiator camphoroquinone, have been found to promote oxidative DNA damage via the generation of reactive oxygen species (ROS) [11].

There is quite limited knowledge concerning the genotoxic and mutagenic effects of the many compounds released in eluates of commercially available, widely used composite resins and dental adhesives [12], [13]. Monitoring the genotoxicity of these eluates would provide a better understanding of their interaction with the oral tissues and the potential to cause genotoxic effects in vivo.

Recently, we have observed that eluates from specific direct composite resins show extensive time- and concentration-dependent cytotoxicity to murine WEHI 13 var fibroblasts, accompanied by induction of DNA fragmentation, as shown by agarose gel electrophoresis [14]. This prompted us to extend these observations in the present study, which was aimed at testing the effect of five widely used composite resins for direct and indirect dental restorations with respect to induction of sister-chromatid exchange (SCE), chromosomal aberrations (CAs), and effects on cell-cycle kinetics and mitotic indices of human peripheral lymphocytes. SCE have been proposed as a very sensitive indicator of DNA damage and/or subsequent DNA repair. In combination with the evaluation of chromosome aberrations, SCE can provide insight in the cytogenetic damage induced by various genotoxic agents at very low concentrations. Moreover, the determination of proliferation rates and mitotic indices in lymphocyte cultures has been proved to be a very useful and sensitive indicator of the cytostatic and cytotoxic action of various environmental hazards or therapeutic agents [15], [16]. Our test hypothesis was that laboratory-polymerized (indirect) composite resins would be less cytotoxic and genotoxic compared with conventional direct composites.

Section snippets

Materials and sample preparation

Two hybrid composites (Tetric Ceram/Ivoclar-Vivadent, Schaan, Lichtenstein and Filtek Z-250/3M ESPE, Seefeld, Germany), one nano-hybrid (Simile/Pentron, Wallingford, USA) and two second-generation laboratory composites for indirect restorations (Adoro/Ivoclar-Vivadent, Schaan, Lichtenstein and Conquest Sculpture/Pentron Wallingford, CT, USA) were tested (Table 1). Specimens of each biomaterial were prepared aseptically with a prefabricated mould as disks of 5-mm diameter and 2-mm thickness,

Results

All eluates from direct composite resins (Tetric Ceram, Simile and Filtek Z-250) induced a statistically significant increase in the frequencies of SCE, accompanied by suppression of PRI and MI in primary cultures of human lymphocytes, as shown in all three experiments, each using blood from a different donor (Table 2, Table 3, Table 4). The increase in SCE was comparable to that induced by the lower test concentrations of melphalan (i.e. 0.5 and 1.0 μM), a well-known alkylating agent that was

Discussion

According to the data presented in this study, eluates from the three direct composites (Tetric Ceram, Filtek Z-250 and Simile) were genotoxic, since they markedly increased the frequency of SCE in human lymphocyte cultures, up to levels comparable to those induced by 0.5–1.0 μM of melphalan, a well-known alkylating agent that was used as a positive control. The extracts were also clastogenic, given the significant increase in the number of CAs, up to values that were much higher than those

Acknowledgements

Dr. A.A. Bakopoulou wishes to thank “Alexandros S. Onassis” Public Benefit Foundation in Athens, Greece, for providing her with a post-graduate fellowship.

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