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In vitro Estrogenicity of Resin CompositesDepartment of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; Correspondence: * corresponding author, ebisu{at}dent.osaka-u.ac.jp
Previously, we have reported that sealants incorporating bisphenol A dimethacrylate showed estrogenicity by a reporter gene assay. This study tested the hypothesis that commercial composites, which contain various monomers and additives, exhibit estrogenic activity in vitro. The estrogenic activities of eluates obtained from 24 composites and 18 chemicals identified from the composites tested were examined with the use of the reporter gene assay. Among the 24 composites, 6 products were estrogenic, and among the 18 constituents, 1 photostabilizer, 2-hydroxy-4-methoxy-benzophenone (HMBP), 1 photoinitiator, 2,2-dimethoxy-2-phenyl-acetophenone (DMPA), and 1 inhibitor, 2,6-di-tert-butyl-p-cresol (BHT) had significant estrogenic activity. The concentration of HMBP in 4 estrogenic eluates was greater than the minimum concentration required for estrogenicity, and DMPA was found at a higher level than the minimum estrogenic concentration in the remaining 2 estrogenic specimens. These results suggest that the observed estrogenic activity of 6 composites is associated with the elution of either HMBP or DMPA.
Key Words: estrogenicity • resin composites • reporter gene assay • 2-hydroxy-4-methoxy-benzophenone • 2,2-dimethoxy-2-phenyl-acetophenone
There is evidence that abnormalities in the reproductive system of wildlife are caused by certain synthetic chemicals released into the environment. It is probable that similar abnormalities occur in humans (Colborn et al., 1996). These hazardous substances, that have mainly estrogenic activity, are termed endocrine-disrupting chemicals (EDCs). Currently, the adverse effects of EDCs on humans are poorly understood, and it is important to identify potential EDCs among the huge numbers of chemicals and to construct a risk assessment of these chemicals. Previously, we examined the estrogenic activity of dental sealants using a reporter gene assay, and found that a hydrophobic monomer, bisphenol A dimethacrylate (BPA-DMA), was associated with the estrogenic properties of these commercial products (Tarumi et al., 2000). However, the estrogenicity of many other proprietary composites, as well as that of their constituents, remains to be investigatedd. Since commercial composites contain a wide variety of different monomers and additives (Spahl et al., 1994), we hypothesized that several constituents of the composites other than BPA-DMA may have estrogenic activity, and that these compounds may be released after polymerization of the material. The objective of this study was to investigate the estrogenic activity of commercial composites by means of a reporter gene assay, and to determine which constituents had estrogenic activity.
Composite Disks Twenty-four commercially available resin composites were tested (Table 1  ). Each composite paste was placed into a steel mold (5 mm diameter, 1.5 mm high), and the surfaces were covered with celluloid matrix (GC, Tokyo, Japan) and glass slides. The top and bottom surfaces were irradiated for 40 sec each with a light-activation unit (Optilux 501, Demetron/Kerr, Orange, CA, USA).
Monomers and Additives By means of gas chromatography/mass spectrometry (QP5050, Shimadzu, Kyoto, Japan), 18 chemicals—including 5 monomers, 2 photoinitiators, 3 photostabilizers, 2 inhibitors, and 6 co-initiators—were identified as constituents of the 24 composites tested. These chemicals were used in the present study without further purification (Table 2 ).
Each chemical was dissolved in dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries, Osaka, Japan), with the concentration being adjusted from 100 nmol/L to 100 µmol/L with phenol red-free Dulbecco’s-Minimum Essential Medium (D-MEM, Nikken BioMedical Laboratory, Kyoto, Japan) containing 10% charcoal/dextran-treated fetal bovine serum (FBS, HyClone, Logan, UT, USA). The final concentration of DMSO in the samples was 0.1%.
Cell Culture and Transient Transfection
We constructed a reporter plasmid, pGV-B2-4xERE-tk, by inserting 4 repeats of synthetic oligonucleotide for the estrogen-responsive element (ERE) from the Xenopus vitellogenin gene upstream of the herpes simplex viral thymidine kinase gene promoter, linked to the firefly luciferase gene (Toyo Ink, Tokyo, Japan). An expression plasmid for human estrogen receptor A 10-mL cell suspension with the number of cells adjusted to 5.0 x 104/mL was plated on a 90-mm culture dish and incubated for 24 hrs. Transient transfection of the plasmids pGV-B2-4xERE-tk, pEF-LacZ, and HEGO was initiated by means of the FuGENE 6 transfection reagent (Roche Diagnostics, Basel, Switzerland) as described in the manufacturer’s protocol. In addition, a reporter plasmid without ERE, pGV-B2-tk, was also transfected as the negative control for the reporter plasmid used in the post-screening assay.
Reporter Gene Assay After transient transfection, the cells were stripped, divided among the wells of a 12-well culture plate, and incubated for 6 hrs. The medium in each well was aspirated and replaced with a 1.0-mL aliquot of the samples, followed by incubation for a further 6 hrs. The cells then underwent lysis by the addition of a lysis buffer (Pica Gene Luminescence Kit, Toyo Ink). The luciferase activities were measured by a luminometer (EG&G Berthold, Wildbad, Germany), and β-galactosidase activity, the internal control for transfection efficiency, was determined by the method of Nakajima et al.(1993). Relative luciferase activities were calculated as the mean of 3 replicates of luciferase activity standardized against β-galactosidase activity, divided by the activity of 17β-estradiol at 100 pmol/L. Significant differences between each sample and the negative control for D-MEM, or D-MEM with DMSO, were analyzed by ANOVA and Scheffé’s F test, with the level of significance being set at p < 0.05. To confirm the dependence of the activity on ERE, we carried out further experiments on samples with significantly greater luciferase activity than the negative control.
HPLC Analysis
Estrogenic Activities of Cured Composite Disks Of the 24 composites, 6 samples—Charisma (CH), Flowline (FL), Esthet X (EX), Surefil (SF), Spectrum (SP), and Revolution (RE)—showed significantly greater relative luciferase activity than the negative controls (p < 0.05, Fig. 1 ). No significant difference was observed between these samples and the negative controls in the post-screening assay using the control plasmid without ERE (data not shown)..
Estrogenic Activities of Monomers and Additives Among the 18 chemicals, 3 compounds (HMBP, DMPA, and BHT) demonstrated significant luciferase activity (p < 0.05). HMBP and DMPA showed greater relative luciferase activity than the negative controls at 1 µmol/L and over (Figs. 2a , 2b). BHT showed significant differences from the controls at 50 µmol/L and over (Fig. 2c). In the post-screening assay, none of these compounds demonstrated significant differences in comparison with the negative controls (data not shown). The minimum estrogenic concentration of BPA was 100 nmol/L (Fig. 2d).
Concentrations of Components Eluted HMBP was detected in 12 of the 24 eluates at concentrations ranging from 0.02 to 0.35 mg/L (Fig. 3a ), with the concentrations in 4 composites (RE, SP, SF, and EX) exceeding the minimum estrogenic concentration of HMBP determined (1 µmol/L = 0.23 mg/L). DMPA leached from CH and FL at 0.31 and 0.28 mg/L, respectively (Fig. 3b), and these concentrations were greater than those of DMPA required to show estrogenicity (1 µmol/L = 0.26 mg/L). Neither BHT nor BPA was detected in any of the samples.
Recently, considerable attention has been focused on the estrogenicity of chemicals, since several simple tests for screening estrogenic activity have been developed. These methods include competitive ligand-binding (Ireland et al., 1980), cell proliferation (Soto et al., 1995), yeast two-hybrid (Nishikawa et al., 1999), and reporter gene assays. The former three methods have limitations that minimize their usefulness for screening estrogenicity. For example, the competitive binding assay for the estrogen receptor does not allow substances to initiate gene expression following binding, while the cell proliferation assay lacks specificity, since mitogens other than estrogens are also able to influence cell proliferation. The yeast two-hybrid assay has the drawback that permeability of yeast cell walls to these compounds is different from cell membranes (Gray et al., 1997). Therefore, we used the reporter gene assay, which has better specificity and sensitivity than the other methods (Baker, 2001). When the estrogenic activity of the eluates from the cured composite was examined, 6 samples (CH, FL, EX, SF, SP, and RE) exhibited significantly greater luciferase activity than the negative controls. The results of the post-screening assay confirmed that the estrogenicity of the 6 samples was ERE-dependent. Since all 6 composites are based on Bis-GMA, it is possible that the estrogenic activity was the result of elution of BPA. However, HPLC failed to detect any BPA in any of the eluates. The detectable limit of BPA of 1 nmol/L was considerably less than the minimum concentration required for estrogenic activity (100 nmol/L), and it is apparent that BPA is not the cause of the estrogenic activity in any of the 6 eluates.
Our analyses also demonstrated that 3 of the 18 constituents of the composites—photostabilizer HMBP, photoinitiator DMPA, and inhibitor BHT—had estrogenic activity. The minimum concentration of HMBP and DMPA required for estrogenicity was determined to be 1 µmol/L, and both chemicals have estrogenic activity approximately 10 times weaker than that of BPA (Fig. 2 Camphorquinone is used as the photoinitiating compound in most composites, and DMPA was found only in products by Hereaus Kulzer, such as CH and FL. Elution of HMBP from either CH or FL was less than the minimum estrogenic concentration, but DMPA was detected at levels greater than the estrogenic threshold of 1 µmol/L (0.26 mg/L) determined. These results suggest that the estrogenic activity of RE, SP, SF, and EX was associated with the elution of HMBP, while the activity of CH and FL was attributable to the leaching of DMPA. Our series of experiments showed that BHT was not responsible for estrogenic activity, since this compound was not detected in any of the samples. HMBP is a principal component of many UV screening agents and cosmetics (Lewerenz et al., 1972). The studies involving male mice demonstrated that the administration of HMBP for 13 wks at dosages as high as 400 mg/kg/day had no toxic reproductive potential (Daston et al., 1993). However, it has been reported that dermal application of this compound to immature female rats at 1500 mg/kg/day for 4 days caused an increase of approximately 20% in uterine weight (Schlumpf et al., 2001), and HMBP is listed by the Japanese Environment Agency (1998) among "chemicals suspected of having endocrine-disrupting effects". The quantity of HMBP leached from composites in the present study was far less than the amount needed to show estrogenicity in vivo, indicating that the risk of potentially adverse effects in humans is possibly negligible in a clinical situation. The estrogenic effect of DMPA in humans is also considered to be negligible, since the reporter gene assay demonstrated similar estrogenic activity for DMPA and HMBP. In addition, the estrogenic compound BPA was found in saliva in considerably lower amounts only immediately after the placement of sealants, so it is suggested that there seems to be no reason for concern about clinical implications of the estrogenicity of dental filling materials (Arenholt-Bindslev et al., 1999). When one takes these facts into account, the clinical relevance of the results of the present in vitro study remains unclear, and further investigation into the in vivo estrogenic effects of composites is necessary, although the release of estrogenic compounds from composites is basically undesirable.
We thank Prof. Masatoshi Matsuo (Collaborative Research Center for Advanced Science and Technology, Osaka University) for supervising the experiments. This work was supported by Grants-in-Aid for Scientific Research (12557160, 13671998, 14207080) from the Japan Society for the Promotion of Science and by institutional funds. Received for publication March 12, 2003. Revision received September 30, 2003. Accepted for publication January 14, 2004.
Journal of Dental Research, Vol. 83, No. 3,
222-226 (2004) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
, HEGO, provided by Prof. P. Chambon (INSERM, Strasbourg, France), and an expression plasmid encoding β-galactosidase, pEF-LacZ, provided by Prof. Hirano, were also used. 
