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 »  Home  »  Endodontic Articles 7  »  The effect of AH 26 and AH Plus on MCF-7 breast cancer cell proliferation in vitro
The effect of AH 26 and AH Plus on MCF-7 breast cancer cell proliferation in vitro
Introduction - Materials and methods.



R. Pulgar, J. J. Segura-Egea, M. F. Fernandez, A. Serna3 & N. Olea
Department of Stomatology, School of Dentistry, University of Granada, Granada, Spain.
Department of Stomatology, School of Dentistry, University of Sevilla, Sevilla, Spain.
Laboratory of Medical Investigation, Department of Radiology, School of Medicine, University of Granada, Granada, Spain.


Introduction.
AH 26 and AH Plus (Dentsply DeTrey, Konstanz, Germany) are two of the most commonly used root canals sealers, both are epoxy resin-based materials. According to the manufacturer, AH Plus will not release formaldehyde, although AH 26 does (Huang et al.2000). The potential leakage of endodontic sealers into periapical tissues during root canal filling requires that these materials are neither cytotoxic, genotoxic, mutagenic nor associated with any other negative biologic effects. The cytotoxicity, genotoxicity, and mutagenicity of the epoxy resin-based root canal sealers have been studied in vitro by means of several tests. Miletic et al. (2000) reported the high cytotoxicity of AH Plus and AH 26 after determining the number of viable cells using a light microscopy. Cohen et al. (2000) demonstrated the cytotoxic effect of bothAH26andAHPlususing the agar diffusion test. Similar results were obtained by Huang et al. (2000) in cultures of rat hepatocytes.The cytotoxicity of AH Plus when tested using an in vitro culture of human gingival fibroblasts was no longer detectable after 4 h, whereas the cytotoxic effect ofAH26remained at a high level until 5 weeks (Azar et al. 2000). Both AH 26 and AH Plus have been showed to be mutagenic toward strains TA of Salmonella immediately after mixing until 1month after polymerization (Jukic et al. 2000). On the contrary, studies using the growth inhibition test with primary human periodontal ligament fibroblasts and permanent 3T3 monolayers, the procaryotic umu test, the eucaryotic DNA synthesis inhibition test, and the Ames test, did not find cytotoxicity o rmutagenicity with AH Plus (Leyhausen et al.1999).
The monomer 2,2-bis[4-(2-hydroxy-3-methacrylyloxypropoxy) phenyl]-propane (bis GMA), prepared from bisphenol A (BPA) and glycidyl methacrylate (GMA), is the major ingredient of the epoxy-resin based root canal sealers AH 26 and AH Plus (Peutzfeldt 1997). Furthermore, diglycidyl ether of bisphenol A (BADGE) is also an ingredient of AH 26 (Leonardo 1994). The potential impact that the BPA may have on the biocompatibility of endodontic sealers with oral tissues has been of great concern. Pulp studies have shown lack of significant pulpal irritation after the placement of properly sealed resin composite filling containing BPA (Cox et al.1987). Regeneration of a part of the periodontal ligament has been observed in periapical tissue of rat molars after the application of light-cured composite resin as retrofillings (Maeda et al. 1999). Furthermore, root-end sealing of mandibular molars with dentine-bonded resin composite containing BPA showed complete apical healing in 92%of cases (Rudet al. 2001). On the contrary, it has been demonstrated that the resin composites used as retrograde root filling materials reduce the proliferation of gingival fibroblasts and rat sarcoma cells (Peltola et al. 1992).Moreover, it has been shown that BPA, at low concentrations, increased spleen cell proliferation to concanavalin A (Jontell et al. 1995) and inhibits macrophage adhesion to plastic surfaces (Segura et al.1999).
Olea et al. (1996) showed the oestrogenic effect of BPA and BADGE in the MCF-7 breast cancer proliferation assay, and this was confirmed by Hashimoto & Nakamura (2000).Thus, the use of epoxy resin-based materials has been questioned on the basis that bisGMA based composites and sealants leak oestrogenic monomers, such as BPA and BADGE, into the environment (Pulgar et al. 2000).
However, no study has been performed to analyze the potential oestrogenicity of epoxy resin-based endodontic sealers. In this paper the oestrogenic activity of AH 26 and AH Plus are investigated using theMCF-7 based proliferation bioassay (Villalobos et al.1995).

Materials and methods.

Sample preparation.
Six samples of AH 26 and AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany; lot nos. 9706001368 and 9907000915, respectively) were prepared as follows:
  1. paste A - AH Plus (epoxy resin and others);
  2. paste B –AH Plus (amine and others);
  3. mixed AH Plus pastes A/B1:1;
  4. paste AH26;
  5. powder AH26; and
  6. mixed AH 26 paste/powder 1:1.
To prepare samples, 50 mg of each one of the six products were dissolved to saturation in 5 mL ethanol. Then, dilutions (1/100; 1/1000; 1/ 10 000; 1/100 000; and 1/1000 000) were prepared in ethanol and assayed in the proliferation assay.

Cell line and culture conditions.
ClonedMCF-7 human breast cancer cells were grown for routine maintenance in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 5% foetal bovine serum (FBS, BioWithaker, Brussels, Belgium) in an atmosphere of 5% CO2/95% air under saturating humidity at 37 8C.The cells were subcultivated at weekly intervals using a mixture of 0.05% trypsin and 0.01 EDTA (Sigma, St. Louis, MO, USA).

Charcoal-dextran treatment of serum to remove sex steroids.
Plasma-derived human serum was prepared from outdated plasma by adding calcium chloride to a final concentration of 30 mm to facilitate clot formation. Sex steroids were removed from serum by charcoal-dextran stripping. BrieЈy, a suspension of 5% charcoal (Norit A, Sigma Chemical Co, St. Louis, MO, USA) with 0.5% dextran T-70 (Pharmacia-LKB, Uppsala, Sweden) was prepared. Aliquots of the charcoal-dextran suspension of a volume similar to the serum aliquot to be processed were centrifuged at 1000 xg for 10 min Supernatants were aspirated and serum aliquots were mixed with the charcoal pellets. This charcoal-serum mixture was maintained in suspension by rolling at 6 cycles min_1 at 37 8C for 1 h. The suspension was centrifuged at 1000 xg for 20 min, and the supernatant was then filtered through a 0.20-mmfilter (Millipore). Charcoal dextran- treated human serum (CDHuS) was stored at x20 8C until needed.

Cell proliferation experiments.
Estradiol-17a (Sigma, St. Louis, MO, USA) was stored as a 1-mm stock solution in ethanol at _20 8C. The final ethanol concentration in the culture medium did not exceed 0.1%; this concentration did not affect cell yields. MCF-7 cells were used in the test of oestrogenicity according to a technique slightly modified (Villalobos et al. 1995) from that originally described by Soto et al. (1992). BrieЈy, cells were trypsinized and plated in 24-well plates (Limbro, McLean,VA, USA) at initial concentrations of 10 000 cells per well in 5% FBS in DME. Cells were allowed to attach for 24 h, then the seeding medium was replaced with 10% CDHuS-supplemented phenol red-free DME.
Different concentrations of the test compound were added to sample wells, 10 pm estradiol-17b to positive control, and no substance to negative control (hormone- free control cells). The assay was stopped after 144 h by removing medium from wells, fixing the cells and staining them with sulforhodamine-B (SRB). The cells were treated with cold 10% trichloracetic acid and incubated at 4 8C for 30 min, washed five times with tap water and left to dry. Trichloroacetic-fixed cells were stained for 10 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Wells were rinsed with 1% acetic acid and air dried. Bound dye was solubilized with 10 mm Tris base (pH 10.5) in a shaker for 20 min Finally, aliquots were transferred to a 96-well plate and read in a Titertek Multiscan apparatus (Flow, Irvine, CA) at 492 N.M. Linearity of the SRB assay with cell number was verified prior to cell growth experiments. Mean cell numbers from each experiment were normalized to the steroid-free control cultures to correct for differences in the initial seeding density. MCF-7 cell proliferation (fold-over control) was calculated as the ratio between the cell yield obtained with the substance tested (AH 26, AH Plus, or estradiol-17b) and the proliferation of hormone-free control cells (control).

Statistical analysis.
All values were expressed as the mean x SDof four separate experiments performed intriplicate. Mean cell numbers from each experiment were normalized to the steroid-free control cultures to correct for differences in the initial seeding density. Differences between the samples of endodontic sealers and estradiol-17b group were assessed by analysis of variance. A value of P < 0.05 was regarded as statistically significant.