Introduction - Materials and methods.
I. Miletic, S. Jukic, I. Anic, D. Z_eljezic, V. Garaj-Vrhovac, M. Osmak.
School of Dentistry, University of Zagreb, Croatia.
Institute for Medical Research and Occupational Health, Croatia.
The purpose of root canal treatment is to eliminate infection in the root canal and to fill the root canal space. Various commercial sealers have been developed and used for this purpose. One of them, AH26 sealer (Dentsply, DeTrey, Konstanz, Germany), is frequently used because of its excellent sealing ability (Wu et al.1995, Miletic. et al. 1999). It has been demonstrated, however, that the sealer was cytotoxic during setting (Gerosa et al. 1995) which can be, to some extent, explained by the release of formaldehyde (Spangberg et al.1993, Koch1999). A modified version of the material AH Plus (Dentsply) was subsequently developed. According to the manufacturer, AH Plus has better physical and clinical properties than AH26 and the formulation no longer releases formaldehyde.
Root filling materials are usually in close contact with living tissues. Thus, the biological properties of these materials are important as cytotoxic materials can damage periapical tissues, and material with mutagenic potential can induce DNA mutations, possibly causing malignant transformation of the cells (Bertram 2001). Various tests have been developed for determination of mutagenicity. The most commonly used and simplest is the Ames test (Lewis&Chestner1981). However, the positive results of the Ames test alone are not sufficient to estimate the carcinogenic risk to a human population (Cross et al. 1983); rather, these are used to detect potentially dangerous chemicals (Lewis & Chestner 1981). The genotoxicity of dental material should also be investigated using other tests, such as the V79/hprt mutation assay, the micronucleus test and the chromosomal aberration assay (Fenech&Morley1985, Garaj-Vrhovac & Z Keljezic. 2001). The mutagenic effects of AH26 have been determined on rodent cells in vitro using the mutation assay with the mutagenic effect being dependent on the period after mixing (Schweikl et al.1995).
Data on the mutagenic effects ofAH26 and AH Plus on human cells is inconclusive. Therefore, the aim of this study was to determine the cytotoxic and mutagenic effects of AH26 and AH Plus in vitro using a structural chromosomal aberration analysis and micronucleus test.
Materials and methods.
AH Plus and AH26 sealers.
In the present study, two materials were used: AH26 silver free (Dentsply, DeTrey) and AH Plus (Dentsply, DeTrey).The sealers were mixed according to the manufacturerâ€™s instructions in aseptic conditions. In one group of experiments, the mixed material was set for 1 h and crushed; then 0.1 g of the material was eluted with 2mL of dimethyl sulphoxide (DMSO) for1h,24 h and 7 days. These extracts were serially diluted and used for further examination to final concentrations of 1.67, 5.57, 16.7, 55.7 and 167 g mL_1. These were chosen after the concentration of167 g mL_1demonstrated cytotoxicity in the pilot study, so a range of smaller concentration were used to determine doses not causing a significant cytotoxic effect. In the other group of experiments, the material was set for 1 h, 24 h or 7 days in a physiological saline and eluted using the same protocol. The control samples were treated with DMSO diluted in the same way and added to the culture samples.
Chinese hamster V79 cells were grown as a monolayer culture in Dulbeccoâ€™s modified essential medium, which was supplemented with10% foetal calf serum and antibiotics, in a humid atmosphere containing 5%CO2 (Atcc, Global Bioresource Centre, Manassas, VA, USA).
Human lymphocytes were kept at 378C in the F-10 medium (IAEA 1986) in the presence of 0.5 mL phytohaem aglutinine and with 10% of newborn calf serum (Biological Industries, Kibutz, Beit Haemek, Israel).
Chinese hamsterV79 cells were plated in 2 mL growth medium in 24-well plates (7.5 _103 cells per plate). The cellswere incubated for 72 h. There after, they were trypsinized and counted. In parallel samples, the number of viable cells was determined usingnigrosindye. The cytotoxicity was quantified by comparing the number of viable cells in treated samples with the corresponding number of viable cells in the control samples. Each experiment was repeated three times.
To determine the possible mutagenic effect of AH26 and AH Plus, two standard cytogenetic methods were used: the structural chromosomal aberration analysis and the micronucleus test. Both methods were performed on the cultures of human peripheral blood lymphocytes. At the beginning of the procedure, lymphocytes were treated with 5.57, 16.7 and 55.7 g mL_1 of AH26 and AH Plus, all in the range of cytotoxic concentrations (Figs 1and2).The control lymphocytes were treated with DMSO diluted in the same way. For the structural chromosome aberration analysis, the cultures were harvested 48 h after their initiation. Three hours prior to harvesting, 0.2 g mL_1 of colchicine was added. After the slide preparation, 200 metaphases per sample were analysed. For the micronucleus test, 44 h after the culture initiation, 3g mL_1 of cytochalasine B (Sigma, St. Louis, USA) was added. Total time of the cultivation was 72 h. After the slide preparation, 500 binucleated lymphocytes per sample were analysed. The number of chromatid and chromosome breaks and acentric fragments, as well as number of micronuclei was recorded for each sample.
Figure 1. Cytotoxicity of DMSO eluates of mixed AH26 and AH Plus after indicated elution periods.
Figure 2. Cytotoxicity of DMSO eluates ofmixed AH26 and AH Plus after setting in saline solution.
Statistical analysis of cytotoxicity data was performed using the F-test for ratios of the cell number in experimental and control samples, as data had shown no significant in homogeneity of variances. As mutagenic data consisted only of proportion of aberrations and micronuclei, these were evaluated by testing differences between proportions in experimental versus control samples.