TY - JOUR
AU1 - Arossi, G. A.
AU2 - Dihl, R. R.
AU3 - Lehmann, M.
AU4 - Cunha, K. S.
AU5 - Reguly, M. L.
AU6 - de Andrade, Heloísa H. R.
AB - Abstract This in vivo study investigated the genotoxicity of two dental bonding agents: Adper Single Bond Plus and Prime&Bond 2.1. The somatic mutation and recombination test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, as well as point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. This fruit fly has extensive genetic homology to mammals, which makes it a suitable model organism for genotoxic investigations. Adper Single Bond Plus induced statistically significant increases in the frequency of total spots at the highest concentration tested, while Prime&Bond 2.1 was positive at all concentrations tested. The mechanistic basis underlying the genotoxicity of Adper Single Bond Plus relies on mitotic recombination alone, and was different from that of Prime&Bond 2.1, which showed evidence of the contribution of both recombination and mutational events. These findings indicate that both adhesives are inducers of toxic–genetic events, with the mitotic recombination being the main mechanism of action. The clinical significance of these observations has to be interpreted with data obtained in other bioassays. Introduction The development of monomeric resinous materials and adhesive protocols has made it possible to bond the filling material to the tooth structure. The resinous monomers used in dentistry are formed by different organic molecules, such as bisphenol A-glycidyl methacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA) and hydroxyethyl methacrylate (HEMA), which work together as co-polymeric chains (1). These monomers comprise the main organic basis of the majority of composite resins and dental adhesives. Incomplete polymerization of dental resin composites and resin-based bonding agents under clinical conditions results in free resin monomers that may be released from the resin matrix either into the aqueous environment of the oral cavity or into the dentine-pulp system (2–4). Concerning this feature, residual monomers are considered a drawback of composite resins, being associated with cytotoxic effects (5–10). Among the numerous compounds included in dental adhesives, only a few single substances have been identified as genotoxic or mutagenic in vitro. In particular, the monomer TEGDMA might cause serious DNA damage in mammalian cells as indicated by the induction of micronuclei, gene mutation and large DNA sequence deletions in V79 Chinese hamster fibroblasts (11–14). Investigations of the mutagenic effects of clinically used resin-based dental materials at the somatic level are scarce. The micronucleus test has shown that dental adhesive and composite resin aqueous extracts may affect the genome, resulting in chromosomal mutations (15,16). In the present study, we have used the Drosophila wing spot test to evaluate the genotoxicity of two dental bonding agents: Adper Single Bond Plus (Bis-GMA, HEMA and UDMA) and Prime&Bond 2.1 (UDMA and HEMA). This short in vivo test system is based on the loss of heterozygosity in normal genes and the corresponding expression of recessive markers, namely multiple wing hairs (mwh) and flare3 (flr3), in the wing blade of adult flies. The mutational and recombinational potential were quantified as a function of exposure concentration determined for both compounds with special emphasis on their recombinagenic action at two intervals of chromosome 3 of Drosophila melanogaster. Materials and methods Compounds The dental bonding agents tested were Prime&Bond 2.1 (Dentsply DeTrey GmbH, Konstanz, Germany) and Adper Single Bond Plus (3M/ESPE, St Paul, MN, USA), obtained from a regular dental store. Detailed information about the adhesives is shown in Table I. All the concentrations tested for Prime&Bond 2.1 and Adper Single Bond Plus contained 5% of the solvents, represented by acetone and alcohol, respectively. For Adper Single Bond Plus, the highest monomer concentration tested was 5%, while for Prime&Bond 2.1 it was 2%. Negative solvent controls and positive controls (cisplatin 0.05 mM—CAS 15663-27-1—Plastinine®, Pfizer Ltda., São Paulo, Brazil) were also included. Table I Information about the commercially available dental bonding agents Compound  Manufacturer  Monomer composition  Solvent composition  Adper Single Bond Plusa  3M/ESPE  Bis-GMA (10–20%), HEMA (5–15%) and UDMA (1–5%)  Water/alcohol  Prime&Bond 2.1b  Dentsply  UDMA (<20%) and HEMA (10–20%)  Acetone  Compound  Manufacturer  Monomer composition  Solvent composition  Adper Single Bond Plusa  3M/ESPE  Bis-GMA (10–20%), HEMA (5–15%) and UDMA (1–5%)  Water/alcohol  Prime&Bond 2.1b  Dentsply  UDMA (<20%) and HEMA (10–20%)  Acetone  Data source (17): a Adper Single Bond Plus: http://www3.3m.com/search/br/pt002/msdssearchform.do. b Prime&Bond 2.1: http://www.dentsply.com.br. View Large Standard Cross The standard cross was used, flr3 virgin females crossed to mwh males (18), which originated larvae with two genotypic constitutions: (i) mwh+/+flr3—trans-heterozygous for the recessive markers mwh and flr3 and (ii) mwh +/TM3, BdS—heterozygous for balancer chromosome TM3. The induction of genotoxicity in the marker-heterozygous flies produces two mutant clones: (i) single spots, either mwh or flr3, resulting from point or chromosome mutations as well as mitotic recombination (between mwh and flr3) and (ii) twin spots, consisting of both mwh and flr3 subclones, which originate exclusively from mitotic recombination (between flr3 and the centromere) (19). In the balancer-heterozygous genotype, mwh spots reflect predominantly somatic point mutation and chromosome mutation, since mitotic recombination involving the balancer chromosome and its structurally normal homologue is a lethal event (20). Treatments Virgin females of the flr3/In (3LR) TM3, Bds strain were mated to mwh males, as previously described (21). Eggs from this cross were collected during 8-h periods in culture bottles containing standard medium. The resulting 3-day-old larvae were then placed in plastic vials containing 5.0 g of Drosophila Instant Medium (Carolina Biological Supply, Burlington, NC, USA) prepared with the different concentrations of Adper Single Bond Plus (2.5, 3.75 and 5.0%), and Prime&Bond 2.1 (1.0, 1.5 and 2.0%), which were non-toxic. The larvae were fed on this medium until pupation. The surviving adults were collected from the treatment vials and were stored in 70% ethanol. Afterwards, their wings were removed and mounted in Faure's solution on slides. The wings were scored at 400 times magnification for the presence of spots. In each series, 30 individuals were scored. Scoring of flies and data evaluation were conducted following the standard procedures for the wing spot assay, as used in recent investigations (22). Statistical analysis The conditional binomial test of Kastenbaun and Bowman (23) was used to assess differences between the frequencies of each type of spot in treated and concurrent negative control flies. The formulation of two alternative hypotheses allows to distinguish among the possibilities of a positive, weakly positive, inconclusive or negative result of an experiment (24). In the null hypothesis (H0), one assumes that there is no difference in the mutation frequency between the control and treated series. Rejection of the null hypothesis indicates that the treatment resulted in a statistically increased mutation frequency. The alternative hypothesis (HA) postulates a priori that the treatment results in an increased mutation frequency compared to the spontaneous frequency. This alternative hypothesis is rejected if the observed mutation frequency is significantly lower than the postulated increased frequency. Rejection indicates that the treatment did not produce the increase required to consider the compound as mutagenic. If neither of the two hypotheses is rejected, the results are considered inconclusive, as one cannot accept at the same time the two mutually exclusive hypotheses. In the practical application of the decision procedure, one defines a specific alternative hypothesis requiring that the mutation frequency in the treated series is at least m (multiplication factor) times greater than in the control series, which is then used together with the null hypothesis. Since small single spots and total spots have a comparatively high spontaneous frequency, m is fixed at a value of 2 (testing for a doubling of the spontaneous frequency). For the large single spots and the twin spots, which have a low spontaneous frequency, m = 5 is used. It may happen in this case that both hypotheses have to be rejected. This would mean that the treatment is weakly mutagenic, but leads to a mutation frequency that is significantly lower than m times the control frequency (25). Results The results obtained from the two experiments testing the genotoxicity of Prime&Bond 2.1 and Adper Single Bond Plus in the wing spot assay are summarized in Table II. These compounds were supplied to 72-h-old larvae (third instar) at concentrations ranging from 2.5 to 5.0% for Adper Single Bond Plus and from 1.0 to 2.0% for Prime&Bond 2.1. Since there were no significant differences in the responses in the two experiments, the data were pooled. Negative solvent controls, as well as a positive control (cisplatin 0.05 mM), were included in each experiment. The data for the different mutant spots are given for both mwh/flr3 and mwh/TM3 progenies. For purposes of statistical evaluation, the results from flies treated with the test agents were compared with the data from the corresponding negative controls. Non-toxic concentrations were used to perform the genotoxic evaluation. In this study, the negative control frequencies found (0.57 and 1.0) were in agreement with the normal background range observed in our laboratory and are in accordance with previous results reported by other authors (26,27). Table II Fly spot data obtained after exposure of marker (mwh/flr3) and balancer (mwh/TM3) heterozygous larvae of Drosophila melanogaster to Adper Single Bond Plus (single bond) and Prime&Bond 2.1 on the standard cross in the SMART test Genotypes  Dilutions (% of monomer)  No. of flies (n)  Spots per fly (no. of spots)/statistical diagnosisa   Total mwh clonesc (n)  Small single spotsb (1–2 cells) (m = 2)  Large single spotsb (>2 cells) (m = 5)  Twin spots (m = 5)  Total spots (m = 2)    Single bond              mwh/flr3  Alcohol 5%  30  0.93 (28)  0.07 (02)  0.00 (00)  1.00 (30)  30    2.50  30  0.97 (29) −  0.07 (02) i  0.03 (01) i  1.07 (32) −  32    3.75  30  1.07 (32) −  0.10 (03) i  0.03 (01) i  1.20 (36) −  36    5.00  30  1.43 (43) +  0.20 (06) i  0.00 (00) i  1.63 (49) +  49    Cisplatin 0.05 mM  30  19.17 (575) +  10.77 (323) +  2.70 (81) +  32.63 (979) +  944  mwh/TM3  Alcohol 5%  30  0.50 (15)  0.00 (00)  d  0.50 (15)  15    2.50  30  0.37 (11) −  0.13 (04) i    0.50 (15) −  15    3.75  30  0.37 (11) −  0.00 (00) i    0.37 (11) −  11    5.00  30  0.30 (09) −  0.07 (02) i    0.37 (11) −  11    Cisplatin 0.05 mM  30  5.93 (178) +  1.97 (59) +    7.90 (237) +  237    Prime&Bond 2.1              mwh/flr3  Acetone 5%  30  0.40 (12)  0.10 (03)  0.07 (02)  0.57 (17)  17    1.00  30  0.87 (26) +  0.07 (02) i  0.07 (02) i  1.00 (30) +  30    1.50  30  1.00 (30) +  0.07 (02) i  0.27 (08) i  1.33 (40) +  40    2.00  30  0.87 (26) +  0.40 (12) +  0.13 (04) i  1.40 (42) +  42    Cisplatin 0.05 mM  30  22.20 (666) +  10.43 (313) +  2.73 (82) +  35.37 (1061) +  1061  mwh/TM3  Acetone 5%  30  0.37 (11)  0.07 (02)  d  0.43 (13)  13    1.00  30  0.37 (11) i  0.00 (00) i    0.37 (11) −  11    1.50  30  0.30 (09) −  0.10 (03) i    0.40 (12) −  12    2.00  30  0.77 (23) +  0.03 (01) i    0.80 (24) +  24    Cisplatin 0.05 mM  30  4.23 (127) +  1.63 (49) +    5.87 (176) +  176  Genotypes  Dilutions (% of monomer)  No. of flies (n)  Spots per fly (no. of spots)/statistical diagnosisa   Total mwh clonesc (n)  Small single spotsb (1–2 cells) (m = 2)  Large single spotsb (>2 cells) (m = 5)  Twin spots (m = 5)  Total spots (m = 2)    Single bond              mwh/flr3  Alcohol 5%  30  0.93 (28)  0.07 (02)  0.00 (00)  1.00 (30)  30    2.50  30  0.97 (29) −  0.07 (02) i  0.03 (01) i  1.07 (32) −  32    3.75  30  1.07 (32) −  0.10 (03) i  0.03 (01) i  1.20 (36) −  36    5.00  30  1.43 (43) +  0.20 (06) i  0.00 (00) i  1.63 (49) +  49    Cisplatin 0.05 mM  30  19.17 (575) +  10.77 (323) +  2.70 (81) +  32.63 (979) +  944  mwh/TM3  Alcohol 5%  30  0.50 (15)  0.00 (00)  d  0.50 (15)  15    2.50  30  0.37 (11) −  0.13 (04) i    0.50 (15) −  15    3.75  30  0.37 (11) −  0.00 (00) i    0.37 (11) −  11    5.00  30  0.30 (09) −  0.07 (02) i    0.37 (11) −  11    Cisplatin 0.05 mM  30  5.93 (178) +  1.97 (59) +    7.90 (237) +  237    Prime&Bond 2.1              mwh/flr3  Acetone 5%  30  0.40 (12)  0.10 (03)  0.07 (02)  0.57 (17)  17    1.00  30  0.87 (26) +  0.07 (02) i  0.07 (02) i  1.00 (30) +  30    1.50  30  1.00 (30) +  0.07 (02) i  0.27 (08) i  1.33 (40) +  40    2.00  30  0.87 (26) +  0.40 (12) +  0.13 (04) i  1.40 (42) +  42    Cisplatin 0.05 mM  30  22.20 (666) +  10.43 (313) +  2.73 (82) +  35.37 (1061) +  1061  mwh/TM3  Acetone 5%  30  0.37 (11)  0.07 (02)  d  0.43 (13)  13    1.00  30  0.37 (11) i  0.00 (00) i    0.37 (11) −  11    1.50  30  0.30 (09) −  0.10 (03) i    0.40 (12) −  12    2.00  30  0.77 (23) +  0.03 (01) i    0.80 (24) +  24    Cisplatin 0.05 mM  30  4.23 (127) +  1.63 (49) +    5.87 (176) +  176  a Statistical diagnoses according to Frei and Würgler (24): +, positive; −, negative; i, inconclusive; and m, multiplication factor for the assessment of significantly negative results. Significance levels α = β= 0.05. b Including rare flr3 spots. c Considering mwh clones from mwh single spots and from twin spots. d Only mwh single spots can be observed in mwh/TM3 heterozygotes as the balancer chromosome TM3 does not carry the flr3 mutation. View Large In the mwh/flr3 genotype, the Prime&Bond 2.1 genotoxicity figures showed significant increases in the total of spots for the three concentrations analysed. The test agent gave inconclusive responses for large single spots at the 1.0 and 1.5 concentrations, as well as for twin clones at all doses tested. In balancer-heterozygous flies (mwh/TM3), a statistically significant response was observed only at the highest concentration, indicating that it induces both recombinagenic and mutational events. In the wings of marker-heterozygous flies (mwh/flr3), Adper Single Bond Plus promoted an increase in the total spot frequency only at the highest dose, but had no effect in the balancer-heterozygous flies (mwh/TM3), which express only gene and chromosomal mutation. This result suggested that its action is related to the induction of homologous recombination (HR)—the main event picked up by the somatic mutation and recombination test (SMART). However, Prime&Bond 2.1 at a concentration of 2% induced an increase in the total spot frequency in both genotypes analysed (mwh/flr3 and mwh/TM3), which means it is able to induce both mutagenic and recombinagenic events. Discussion Investigations of different types of dental adhesives have been performed using V79 and fibroblast cell cultures. Demirci et al. (16) studied Clearfil SE bond, Clearfil Protect bond, Prompt L-Pop, AdheSE and Excite and showed that AdheSE was the only adhesive to increase micronucleus frequency in cultured V79 cells, by up to 6-fold when compared to untreated control cells. In contrast, chromosome and chromatid breaks, as well as acentric fragments, were induced in human lymphocytes by OptiBond Solo Plus, Excite, Adper Single Bond 2 and Adper Single Bond (28). Our positive results with the Drosophila wing spot test show that Adper Single Bond Plus promoted HR, although Prime&Bond 2.1 induced recombinagenic and, to a lesser extent, mutational events. These results suggest that the dental adhesives investigated produce primary DNA damage that is to a considerable extent further processed by recombinational DNA repair pathways. According to the manufacturer, the monomer composition present in Adper Single Bond Plus included Bis-GMA (10–20%), HEMA (5–15%) and UDMA (1–5%)—although only UDMA (<20%) and HEMA (10–20%) are present in the formula of Prime&Bond 2.1. Both compounds—HEMA and UDMA—have been identified as cytotoxic agents by a variety of different methods, all demonstrating changes in basic cell structures such as cell membrane integrity and cell functions like enzyme activities or the synthesis of macromolecules (29). The various biological effects associated with UDMA and HEMA include activities that target cellular DNA. The induction of chromosomal lesions by these substances has been demonstrated in vitro (13,30). Initiation of DNA strand breaks by HEMA has been observed in the comet assay (14,31). Concerning Bis-GMA mutagenesis, the literature data are limited to in vitro studies demonstrating that it did not present significant activity as inducer of point and chromosome mutations (11,13,30). By using SMART, our research group has previously provided evidence that the mechanistic basis underlying the genotoxicity of UDMA is related to HR and gene/chromosomal mutation. Despite the fact that SMART is an extremely precise tool to pick up genotoxic agents, negative results concerning Bis-GMA and HEMA were observed—suggesting that neither substance can cause DNA damage (G. A. Arossi et al., in preparation). By associating this observation with the data of the present study, we could attribute the genotoxicity of both Prime&Bond 2.1 and Adper Single Bond Plus to the presence of UDMA. Therefore, the higher potency observed for Prime&Bond 2.1 might be related to the higher UDMA concentration (<20%) present in the composition of this adhesive—in relation to Adper Single Bond Plus (1–5%). However, UDMA genotoxic activity was associated with the induction of recombination and mutation, while for both dental adhesives HR was the prevalent genotoxic event. This differential pattern might be related to interactions between the other resin monomers present in their formulae. The literature describes dentin bonding agents as inducers of chromosomal mutation in vitro. The evidence indicating that the major effect observed in our study is an increased frequency of mitotic recombination emphasizes another hazard that could be associated to dental adhesives—the increase in HR. Although HR is an important pathway of DNA repair, there is growing evidence that deleterious genomic rearrangements may result from HR, which means that HR events may play a causative role in carcinogenesis. Increased HR frequencies have been found in cancer cells and cancer-prone hereditary human disorders distinguished by mutations in genes playing a role in HR. 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TI - In vivo genotoxicity of dental bonding agents
JF - Mutagenesis
DO - 10.1093/mutage/gen066
DA - 2008-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/in-vivo-genotoxicity-of-dental-bonding-agents-r8JO4Ljo5O
SP - 169
EP - 172
VL - 24
IS - 2
DP - DeepDyve
ER -