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RESEARCH REPORT |
1 Department of Operative Dentistry and Periodontology, University of Regensburg, D-93042 Regensburg, Germany; and
2 3M ESPE AG, D-82229 Seefeld, Germany;
* corresponding author, Helmut.schweikl{at}klinik.uni-regensburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: oxirane • silorane • mutagenicity • V79/HPRT • micronucleus test
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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There are only a few reports on the biological effects of new epoxy-based dental systems. Among others, the compound epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate (CyracureTM UVR-6105, Union Carbide [see Table 1
in Eick et al., 2002, for manufacturer’s information]) tested severely cytotoxic in the agar diffusion assay, but TC50 values determined with a quantitative assay were considerably higher than those determined for Bis-GMA or epoxy compounds like AralditeTM and EponTM. No cytotoxic effects were reported with solid epoxy-based resin composites with acceptable compressive strengths (Kostoryz et al., 1999; Eick et al., 2002). Some findings on biological effects of oxiranes with underlying genetic mechanisms were contradictory. CyracureTM UVR-6105 was reported to test negative in the Ames test in Salmonella tester strain TA100 (Eick et al., 2002). In contrast, we found a weak but dose-related increase of mutant frequencies induced by the same chemical (epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate) (K-126), indicating the induction of gene mutations in a bacterial test system. The compound was even activated by a metabolically active homogenate from rat liver (S9) (Schweikl et al., 2002). We also found inductions of gene mutations by various new oxiranes in Salmonella tester strains TA100 and TA102, but no effects with siloranes except for one compound (Schweikl et al., 2002). However, there is experimental evidence that some reactive epoxides are mostly potent inducers of chromosomal aberrations in cells of higher organisms (Ehrenberg and Hussain, 1981). Therefore, it was likely that some of the bifunctional compounds which we tested earlier in the Ames test might preferentially cause genetic lesions (clastogenic effects or large deletions) not detected by this bacterial gene mutation assay. Here, we present data from two test systems to improve our knowledge on the genetic activities and the types of mutations in mammalian cells induced by reactive oxiranes and siloranes (Waters et al., 1999).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Micronucleus Test in vitro
V79B Chinese hamster lung fibroblast cells were a gift from Prof. G. Speit (University of Ulm, Germany). The cells (1 x 105) were cultivated on microscopic glass slides in 4 mL minimal essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) for 24 hrs at 37°C in an air atmosphere containing 5% CO2. Test compounds were first dissolved in dimethyl sulfoxide (1 mol/L stock solution) and further diluted in cell culture medium following standard protocols (ISO, 1992).
Then, V79B cells were continuously exposed to increasing concentrations of the test chemicals in cell culture medium for 24 hrs (direct exposure; long exposure period). These dilutions contained DMSO concentrations not higher than 1% which tested nontoxic in V79 cells under the current experimental conditions (data not shown). The induction of micronuclei was also analyzed after a short exposure period (4 hrs) in the presence and absence of a metabolically active liver homogenate (S9 fraction) as described in detail elsewhere (Schweikl et al., 2001). At least two independent experiments were performed to show reproducibility of the results, and variability of the micronuclei (MN) rates in one experiment was indicated by mean values and standard deviations (SD) of three independently treated cell cultures per concentration. Micronuclei were analyzed microscopically in 1000 cells per culture (slide). A chemical was considered positive if at least a three-fold increase in the number of micronuclei over negative controls was observed at one or more concentrations (Miller et al., 1997). Ethylmethane sulfonate (EMS) and cyclophosphamide (CP; in the presence of S9) served as positive control substances.
Hypoxanthine Phosphoribosyl Transferase (HPRT) Gene Mutation Assay
V79B cells were routinely cultivated as described above. Test compounds were dissolved in dimethyl sulfoxide (1 mol/L stock solution) and diluted in cell culture medium. Then, the cell cultures were exposed to various concentrations of the test compounds for 4 hrs. The HPRT assay in the presence and absence of a homogenate from rat liver (S9 fraction) was carried out as described in detail elsewhere (Schweikl et al., 1998). The experiments were conducted with one plate per dose for HPRT-deficient mutant isolation, and the experiments were repeated at least once. Mean values of mutant frequencies of two independent experiments are given in Table 2. Ethylmethane sulfonate (EMS) and 9,10-dimethyl-1,2-benzanthracene (DMBA; in the presence of S9) were used as positive control substances (Glatt, 1994).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate (Est-Ep) was less effective, and 0.1 mmol/L diglycidyl ether of bisphenol A (BADGE) induced numbers of micronuclei about seven-fold lower than those caused by Eth-Ep (Table 1). The carboxylate group of Est-Ep was replaced by a methylsubstituted siloxane group to create a silorane of similar structure, di-3,4-epoxy cyclohexylmethyl-dimethyl-silane (DiMe-Sil). DiMe-Sil caused effects comparable with those induced by BADGE. However, no mutagenic effect was detected when one of the methyl groups of DiMe-Sil was substituted by benzene to synthesize methyl-bis[2-(7-oxabicyclo[4.1.0]hept-3-yl)ethyl]phenyl silane (Ph-Sil). Low concentrations of 1,4-bis(2,3-epoxypropyloxypropyl-dimethylsilyl)benzene (Phen-Glyc) were weakly mutagenic. No mutagenic effects were detected with the large silorane, 1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl cyclotetrasiloxane (TET-Sil), in the micronucleus test after direct exposure (Table 1). We observed that genotoxic potencies of the directly (-S9) active compounds Est-Ep, Eth-Ep, BADGE, and DiMe-Sil were reduced by a factor of 2–3 after a short exposure period (4 hrs). Moreover, no induction of micronuclei by any of these chemicals was observed in the presence of a metabolically active S9 fraction (Fig. 2). No elevated numbers of micronuclei were determined with Ph-Sil, Phen-Glyc, and TET-Sil with and without S9 (data not shown).
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). At a concentration of 0.3 mmol/L, Est-Ep induced a seven-fold increase of HPRT-deficient V79 cells after direct testing (-S9). Eth-Ep was about four-fold more effective than Est-Ep at the same concentration. Cytotoxicity of Est-Ep was reduced by S9 as deduced from cell survival in cultures exposed to concentrations higher than 1 mmol/L. However, gene mutations more than 10-fold higher than controls were caused by a concentration of 5 mmol/L Est-Ep. Moreover, Eth-Ep induced mutant frequencies more than 20-fold higher than controls in the presence of S9 (Table 2). No increase of mutant frequencies was determined with all other test compounds (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Our findings with the new chemicals relate to earlier reports on the mutagenicity of epoxides. It has been demonstrated that epoxides are efficient inducers of chromosomal aberrations in vitro and in vivo. Diepoxides in particular may exhibit "radiomimetic" properties (Ehrenberg and Hussain, 1981; Seiler, 1984; Sinsheimer et al., 1993). It was observed previously that the mutagenic activity of monofunctional and difunctional glycidyl compounds varied depending on the genetic endpoints. Monofunctional compounds were more active in inducing gene mutations in Salmonella typhimurium but hardly induced chromosomal breaks in CHO cells. On the other hand, the bifunctional compounds were only weakly active in Salmonella but induced chromosome aberrations (Seiler, 1984). However, bifunctional epoxides like 1,2,3,4-diepoxybutane caused mutations in the hprt gene of human TK6 cells. These gene mutations were associated with increased frequencies of sequence deletions at the 5' region of the hprt gene (Steen et al., 1997).
In the HPRT assay, Eth-Ep and Est-Ep were metabolically modified by a liver homogenate, resulting, first, in reduced cytotoxicity, and, second, in the induction of higher mutation frequencies caused by higher concentrations of the test compounds and their metabolites, respectively. It is remarkable, however, that the types of lesions induced under these experimental conditions were not indicated with the micronucleus test (MNT) in the presence of S9. Moreover, none of the new siloranes was activated to a mutagen detectable in the V79/HPRT assay.
Since the incubation conditions are identical in the HPRT assay and the micronucleus test in the presence of S9, we hypothesize that Eth-Ep is converted to a monofunctional compound (diol epoxide) through hydrolytic ring-opening by an epoxide hydrolase. A high epoxide hydrolase activity was associated with S9 extracted from rat liver (Bentley et al., 1985). The diol epoxide of Eth-Ep might then act as an alkylating agent to induce mostly point mutations in the hprt gene. Since only relatively low mutation frequencies were induced, even at very high concentrations, it is also likely that the diol epoxide of Eth-Ep was further converted to a bis-diol, a metabolite which might be inactive in the induction of mutations. It has been shown that the diol epoxide of BADGE was less potent than BADGE, and no mutations were detected with the bis-diol in the Ames test (Sueiro et al., 2001). The hypothetical considerations of the enzymatic modifications of compounds tested here are based on experimental work with butadiene. This chemical and its metabolites have been used in several studies to clarify the mechanisms of the induction of mutations by monofunctional and bifunctional epoxides. Butadiene is metabolically activated to three mutagenic metabolites (Recio et al., 2001).
Unlike the case of Eth-Ep, we speculate that Est-Ep is first converted to monofunctional compounds, because the ester linkage is cleaved by an esterase activity. Further, the concentrations of the resulting monofunctional epoxides are probably kept at low levels because of the high epoxide hydrolase activity during the four-hour incubation period associated with S9. It is likely that this is the reason for the low mutant frequencies observed at the hprt locus. Thus, the analysis of metabolites of Eth-Ep, Est-Ep, and other compounds which were created by S9 here will shed light on the metabolites with genotoxic activity at the various endpoints used so far in the present investigations and in a recent study (Schweikl et al., 2002).
CyracureTM UVR-6105 was considered non-mutagenic by others, because no effects were detected in S. typhimurium TA100 (Yourtee et al., 2001; Eick et al., 2002). These reports are in contrast to our findings, and the data presented here add further experimental evidence to our recent observations, which characterized Est-Ep as a mutagenic oxirane in S. typhimurium TA100 (Schweikl et al., 2002).
In summary, the mutagenic activities of various newly synthesized oxiranes and siloranes were estimated in mammalian cells here for the first time. Our investigations indicated that Eth-Ep and Est-Ep are clastogenic substances that preferentially induced chromosomal mutations in vitro after direct exposure. The silorane DiMe-Sil, which is structurally related to Eth-Ep and Est-Ep, was less active than these oxiranes, and no effects were detected with Ph-Sil. No induction of gene mutations by siloranes was determined in our recent study, except for weak effects of one compound (Schweikl et al., 2002). From these data, it appears that the mutagenic potential of the various siloranes tested so far in various test systems is much lower than those of related oxiranes. Because of some strong mutagenic effects caused by the reactive oxiranes Est-Ep and Eth-Ep at various genetic endpoints in vitro, these chemicals are now candidate model compounds for detailed in vivo analyses. Considering a complete dental composite based on an organic matrix which contains some of the new monomers analyzed here, the clinical relevance of the data presented will be correlated to parameters like the amounts of residual monomers released from polymerized resins after polymerization, and exposure situations for patients and dental personnel.
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ACKNOWLEDGMENTS |
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Received December 2, 2002; Last revision June 2, 2003; Accepted September 17, 2003
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REFERENCES |
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