Cytotoxicity and mutagenicity of AH26 and AH Plus sealers

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 manufacturer’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.

Cell cultures.
Chinese hamster V79 cells were grown as a monolayer culture in Dulbecco’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).

Cytotoxicity assay.
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.

Mutagenicity assay.
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.

 Results.

As shown in Fig.1, both sealers showed strong cytotoxic effects in concentrations of 55.7 and 167 g mL_1 for all three periods, while for the concentrationof16.7 g mL_1, AH Plus had a significantly stronger cytotoxic effect than AH26 (F-test, P < 0.05 for all three periods). Response curves were similar in samples eluted for different periods of time (F-test, P > 0.05 for both materials and concentrations). 
The cytotoxic effects of set material for different periods of time are shown in Fig. 2.Againthe dose-response curves of cell survival were obtained. AH Plus was again significantly (F-test, P < 0.05) more cytotoxic than AH26 in concentrations of 16.7 g mL_1, for 1 and 24 h setting times and also at 55.7 and 167 g mL_1 for the 7-day period. Further, the cytotoxic effect was shown to be dependent on the setting time. ForAH26, reduced cytotoxicity was found for samples set for 7 days, when compared to 24 and1 h setting times, for concentrations of 16.7, 55.7 and 167 g mL_1 (P < 0.05 for 24 h vs.7 days comparison, as well as for 7 days vs. 1 h comparison). The cytotoxicity of AH Plus was reduced only after 7 days for the concentration of16.7 g mL_1. 
According to these results, the concentration of 5.57, 16.7 and 55.7 g mL_1 of AH26 and AH Plus were used for determination of mutagenic potential. Table 1 shows the results of the structural chromosomal aberration analysis and the micronucleus test. In the cultures treated with AH Plus, chromatid and chromosome breaks and acentric fragments were found. However, there was no significant difference in their numbers between control cells treated with corresponding dilution of DMSO and lymphocytes treatedwithAH26andAHPlus. Also, no significant difference was found regarding the setting time or elution time of the AH Plus (1 h, 24 h and 7 days). Lymphocytes treated with AH26 did not show any significant increase in the number of structural chromosomal aberrations.

Table 1. Results of the structural chromosomal aberration analysis and micronucleus test for AH26 and AH Plus with different concentrations and setting times (1 h, 24 h and 7 days).

The micronucleus test showed results similar to those of the structural aberration analysis. There were no statistical differences between treated and control samples. Only in the blood samples treated with 16.7 g mL_1 AH26 and 16.7 g mL_1 AH Plus that were obtained by 7 days elution in DMSO, there was a slight but insignificant increase in the number of aberrations and micronuclei. In the samples treated with 55.7 g mL_1 AH26 obtained by1 h and 7 days setting period, a statistically insignificant increase in the frequency of micronuclei was found.