According to the above, DMPT, DMAEMA and DMABEE could be cytotoxic, and may have adverse effects on CHO-K1 cells, a cell line popularly used for cytotoxicity and genotoxicity assays. In previous studies, however, still not explain the mechanism of disturbane of cell growth clearly. Moreover, the result may be inconsistent in different investigations about ROS interference, cell cycle analysis and DNA damage assay.
Therefore, the objectives of our study was to investigate (1) whether DMPT, DMAEMA and DMABEE released form commercial resin composites are cytotoxic and may cause inhibition of cell growth on CHO-K1 cells. (2) Whether ROS may be the main component that induces cell apoptosis and DNA damages on CHO-K1 cells after exposing to DMPT, DMAEMA and DMABEE. (3) Whether NAC, catalase and esterase could be effective to reduce or prevent cytotoxicity or genotoxicity. In order to enhance the safety of using DMPT, DMAEMA and DMABEE, the knowledge of the substance released from resin composites is important and essential.
Chapter III. Materials and Methods
3.1 Chemicals
N,N-dimethy-p-toluidine (DMPT), 2-dimethylaminoethyl methacrylate (DMAEMA) , 4-dimethylaminobenzoic acid ethyl ester (DMABEE), dimethyl-sulphoxide (DMSO), propidium iodide, catalase, N-acetyl-cysteine (NAC), esterase and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma / Aldrich (Sigma Chemical Company, St. Louis, MO, USA). Reagents for cytometry flow were obtained from Becton Dickson (Worldwide Inc. Dan-Jose, CA, USA). Cell-culture medium (F-12) and reagents were from Life Technologies (Gibco, Life Technologies, NY, USA). 4',6-diamidino-2-phenylindole (DAPI) and rhodamine phalloidin for immunofluorence were purchased from Life Technologies (Gibco, Life Technologies, NY, USA). DMPT was dissolved in DMSO as stock in 5 M and diluted to various concentrations in culture medium (1.0, 2.5, 5.0, 7.5 and 10.0 mM). DMAEMA was stored in 5 M by dissolving with DMSO and prepared at 0.5, 1.0, 1.5 2.5 and 3.5 mM in culture medium. DMABEE was soluble at 1 M in DMSO and arranged in different concentration in culture medium (0.1, 0.25, 0.5, 0.75, 1.0 mM). The amount of DMSO in each culture well is equal.
3.2 CHO-K1 cells and cell culture
CHO-K1 cells were purchased from ATCC (Queens Road, Teddington, United Kingdom), and cultured in F-12 nutrient mixture medium, and were incubated at 37°C in water-saturated atmosphere containing 95% air and 5% CO2.
3.3 Evaluation of cell growth
To determine the inhibition of CHO-K1 growth by the chemicals (DMPT, DMAEMA and DMABEE), both MTT assay and cell colony assay were used in this study. An equal number of CHO-K1 cells (1.0 × 10⁶ / well) were seeded in 2 ml of F-12 medium in 6-well plate. The cells were incubated overnight at 37°C in a humidified atmosphere of 5% CO2. For each stock solution of 3 chemicals in DMSO, it was diluted in cell culture medium to obtain a final DMSO concentration of 0.2%. The cells were then treated with various concentrations of 3 chemicals in final concentration of (1) 1.0, 2.5, 5.0, 7.5 and 10.0 mM (DMPT), (2) 0.5, 1.0, 1.5 2.5 and 3.5 mM (DMAEMA), (3) 0.1, 0.25, 0.5, 0.75, 1.0 mM (DMABEE).
For short-term effects, MTT assay is used. After the cultured cells were incubated for 24 hours, 20 µl MTT was added to each well and the plates were incubated for two hours at 37°C. Absorbance at 590 nm was measured with a Dynatech Microwell plate reader (Dynatech Labs, Inc, Chantilly, VA, USA), whereby cell proliferation rate was calculated from the absorbance value.
On the other hand, for long-term effect, colony forming cell (CFC) assay has applied.
After the cultured were incubated (200 cells / well) for 24 hours, medium was replaced with F-12 medium containing various chemicals and incubated for 7 days. The colonies were counted (cell numbers > 40) and the ratio was calculated to the control group.
3.4 ROS detection (DCF flow cytometry) / GSH depletion (CMF flow cytometry)
The DCF assay is a method to measure the levels of intracellular ROS. It was first described for micro-plate reader by Wang & Joseph (1999), although the original method had already been discovered in the 60s. Basically, cells were incubated with the pro-fluorescent, lipophilic H2-DCF-DA (dihydrodichlorofluorescein diacetate) which can diffuse through the cell membrane. Inside, the acetate groups are cleaved by cellular esterases so the resulting H2-DCF cannot leave the cells. Reaction with ROS, primarily hydrogen peroxide (H2O2), results in the fluorescent molecule DCF (maximum emission was 530 nm).
Growing 2.5 × 10⁵ CHO-K1 cells in 6-well plate in 2 ml F-12 medium overnight. To add different testing substances to medium and incubate for 24 hours. Then, cells were stained with H2-DCF-DA for 30 minutes and collected by using 10% EDTA-trypsin / PBS and F-12 medium. Each sample was rinsed and resuspended with 250~300 λ PBS on ice. DCF fluorescence was measured at 530 nm using FACSCalibur Flow Cytometry. A total of 10000 cells were analyzed for each sample by Cell Quest software (Becton Dickinson).
CHO-K1 cells (2.5 × 10⁵) were incubated in fresh medium containing DMPT, DMAEMA, DMABEE or DMSO(as negative control) for 24 hours. To evaluate whether the three chemicals may induce ROS production GSH depletion, cells were then stained respectively with CMF-DA for 30 min, detached with trypsin/EDTA, washed with PBS and immediately subjected for flow cytometric analysis.
3.5 Apoptosis and necrosis (PI & Annexin V flow cytometry)
To identify the cells undergoing apoptosis or necrosis, cells were stained with PI and FITC-Annexin V. PI detects cells that have lost their plasma membrane integrity, while Annexin V detects early apoptotic cells based on externalized phosphatidylserine.
Chemicals (DMPT, DMAEMA and DMABEE) of various concentration were added to CHO-K1 cell cultures, which were seeded 2.5 × 10⁶ CHO-K1 cells in 6-well plate overnight, for 24 hours. After incubation, the cells were harvested using EDTA-trypsin and F-12 medium / PBS. The cells were then resuspended in 250 ~ 300 λ binding buffer with 4 µl Annexin V-FITC (Becton Dickson) and 8 µl PI (50 µg / ml) solution. The stained samples were kept on ice and subjected to FACSCalibur Flow Cytometry. FITC fluorescence was collected between 515 and 545 nm, as well as PI fluorescence between 564 and 606 nm. A total of 10000 cells were analyzed for each sample by Cell Quest software (Becton Dickinson).
3.6 Cell cycle (PI flow cytometry)
An equal quantity of 2.5 × 10⁵ CHO-K1 cells were plated in 6-well plate overnight.
They were, then, incubated in different concentrations of DMPT, DMAEMA, and DMABEE (as growth assay) for 24 hours.
Cellular DNA content was determined by flow cytometry as described perviously.
Attached and floating cells were collected by using EDTA-trypsin and F-12 medium / PBS. The cells were fixed for a day in a 70% ice-cold ethanol solution. After washing with PBS, the cells were treated with RNase (2 mg / ml) and stained with PI (40 µg / ml). The PI-elicited fluorescence of individual cells was measured using a FACSCalibur Flow Cytometry with laser excitation at 488 nm. A total of 10000 cells were analyzed for each sample by ModFit and Cell Quest software (Becton Dickinson).
3.7 DNA damage (CBMN assay with IF)
The cytokinesis-block micronucleus assay (CBMN assay) is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. The extent of DNA damage are scored specifically in once-divided binucleated (BN) cells that include micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPBs), a biomarker of DNA mis-repair and/or telomere end-fusions, and nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/
or DNA repair complexes (Fenech, Kirsch-Volders et al. 2011). The assay is being applied successfully for monitoring of in vivo genotoxicity exposure, in vitro
genotoxicity testing and in diverse research fields, as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk.
The 18 × 18 mm slides were placed in 6-well plate after sterilization. 1.0 × 10⁵ CHO-K1 cells were seeded in the plate with 2 ml F-12 medium overnight. Before treating with various concentration of substances (DMPT, DMAEMA and DMABEE) for 24 hours, cells were pre-treated with cytochalasin-B, an inhibitor of microfilament ring assembly required for the completion of cytokinesis, for 1 hours. Attached cells were fixed with formaldehyde for 15 minutes, washed with PBS, and rinsed with 2% PBST (Tween 20 and PBS) for 15 minutes. The slides, then, were picked up and added BSA (0.5%) for 45 minutes. Finally, the cells were stained with a mixed solution (BSA and immuno-fluorescence agents (DAPI : rhodamine phalloidin ~ 1 : 3) for 1 hours. DAPI (blue) and rhodamine phalloidin (red) fluorescence were detected by Olympus IX70 Inverted Microscope. A total of 500 BN cells were calculated for each sample.
3.8 Statistical analysis
All experiments were performed at least four times. Statistical analysis of data was performed using the one-way analysis of variance test considering P values less than 5%
as significant. All statistical analyses were carried out with SPSS 12.0 and Sigma Plot 10.0.
Chapter IV. Results
4.1 Effects of DMPT, DMAEMA and DMABEE on CHO-K1 cells
4.1.1 Growth assay DMPT
As shown in Figure 1.1a, DMPT did induce cytotoxicity and growth inhibition in CHO-K1 cells. At concentration of 1.0, 2.5, 5.0, 7.5, 10.0 mM, DMPT inhibited the growth of CHO-K1 cells by 9 to 22% in 24 hours.
The long-term colony survival of CHO-K1 cells significantly decreased under DMPT treatment. Following 24 hours exposure to DMPT and then observation for a week later, colony formation capacity of CHO-K1 cells was suppressed in a dose-related pattern.
As depicted in Figure 1.1b, 1.0 to 10.0 mM of DMPT decreased the colony numbers by 12% to 67%.
DMAEMA
DMAEMA induced cytotoxicity and reduced viable cells of CHO-K1 around 20%, especially in higher concentrations (2.5 mM and 3.5 mM). As shown in Figure 1.2a, it showed a trend of increase during 0.5 mM to 1.5 mM.
Colony formation assay of CHO-K1 cells under DMAEMA treatment showed an obvious suppression. As depicted in Figure 1.2b, the cytotoxicity was directly displayed at a high concentration (3.5 mM) as well as the percentage of colony numbers were less than 20%.
DMABEE
DMABEE inhibited the growth of CHO-K1 cells in a dose-dependent manner. After 24 hours, cell viability, as show in Figure 1.3a, was obviously reduced about 50% under 0.75 and 1.0 mM.
DMABEE showed the strongest cytotoxicity on CHO-K1 among three chemicals in a 24 hours MTT assay, however, it seemed that there was a moderate effect in the long-term colony survival of CHO-K1. Under treating with DMABEE, the colony numbers decreased from 13% to 73 % and had little variation in high concentration (0.5, 0.75 and 1.0 mM) as shown in Figure 1.3b.
4.1.2 Morphological alteration of CHO-K1 cells DMPT
CHO-K1 had mild morphological differences in appearance when grown in F-12 with DMSO as compared to cells exposed to 10.0 mM DMPT for 24 hours. Cells grown in F-12 medium with DMSO were cuboid to round, producing random, loosely arranged distribution (Mendiaz, Mamounas et al. 1986). Cells exposed to DMPT were only slightly different from those grown in the above media, appearing somewhat more round in shape (Fig. 2.1a & b).
DMAEMA
Compared to control group (with DMSO), CHO-K1 cells, exposed to DMAEMA 3.5 mM, seemed to show no difference in cellular morphology (Fig. 2.2a & b).
DMABEE
In contrast, cells grown in F-12 medium with DMABEE were long, spindle-shaped alternation. We have observed that with high concentration of DMABEE, the cells were fibroblastic in appearance with extended cellular processes (Fig. 2.3a & b).
4.1.3 Annexin V-FITC / PI assay DMPT
We demonstrated the occurrence of DMPT-induced cell death by Annexin V-FITC / PI assay. Flow cytometric analysis with Annexin V-FITC / PI staining showed a 24 hours exposure of CHO-K1 cells to DMPT only slightly fluctuated. The percentages of cells residing in 4 quadrants were not varied in different kinds of concentration of DMPT, namely 2.02% to 3.64% in upper left quadrant (necrotic cells), 1.58% to 3.96% in upper right quadrant (late apoptotic cells) and 0.17% to 0.32 % in lower right quadrant (apoptotic cells) (Fig. 3.1a). However, there were no significant change in the results.
DMAEMA
Similar to DMPT, DMAEMA had no obvious effect on Annexin V-FITC / PI assay of CHO-K1 cells. Compared with control cells at 24 hours, cells exposed to 3.5 mM DMAEMA exhibited mild increasing of necrosis (from 1.35% to 2.27%) and late apoptosis (from 2.88% to 2.93%) (Fig 3.1b).
DMABEE
CHO-K1 cells exposed to DMABEE significantly increased the numbers of necrotic cells (upper left quadrant, from 0.91% to 2.81% with significant difference), resulting in cell death without entering early apoptosis (lower right quadrant, from 0.32% to 0.91%
without significant difference) in a dose-dependent manner. Further, the cells exposed to the highest concentration of DMABEE (1.0 mM), an increasing of the amount of cells resided in late apoptosis quadrant was found. Compared with negative control cells, the percentage of late apoptotic cells increased from 1.89% to 6.27% (p < 0.05) (Fig. 3.1c).
4.1.4 PI assay DMPT
CHO-K1 cells treated with DMPT demonstrated growth arrest. A 24 hour exposure of CHO-K1 to different concentrations of DMPT, however, induced no variation on cell cycle distribution (around 45%) (Fig. 4.1a). There was only an elevation on Sub-G0/G1 phase of 10.0 mM DMPT (3.3%), but it did not show significant difference (Fig. 4.2a).
DMAEMA
After treating with DMAEMA, cell cycle of CHO-K1 cells showed a mild increase S phase from 43.9% to 63.7% (p < 0.05), and an opposite result was observed on G0/G1 phase (from 42.3% to 26.4% with significant difference) (Fig. 4.1b). Exposure of CHO-K1 cells to 0.5 to 2.5 mM of DMAEMA showed no significant effect on Sub-G0/G1 phase (around 2%). Following exposure to 3.5 mM of DMAEMA for 24 hours, a
dramatic Sub-G0/G1 phase arrest was noted as revealed by increasing percentage to 5.54% (p < 0.05) about 2 times of the control group (Fig. 4.2b).
DMABEE
When CHO-K1 cells were incubated with DMABEE for 24 hours, it showed a significant effect on cell cycle progression. A marked G0/G1 phase arrest (from 46.9%
to 70.3%) was found from 0.25 mM to 0.75 mM DMABEE. In contrary, the percentage of cells of S phase decreased to 11.63% (p < 0.05) in high concentration of DMABEE (Fig. 4.1c). Sub-G0/G1 phase of CHO-K1 cells exposed to DMABEE displayed a smooth elevation and an significant rising was noted at 1.0 mM of DMABEE (12.5%, p
< 0.05) (Fig. 4.2c).
4.1.5 DCF assay & CMF assay DMPT
A twenty-four hour exposure to 1.0 mM to 10.0 mM DMPT did not affect intracellular ROS content of CHO-K1 cells significantly. The means of fluorescence of CHO-K1 were shown in Figure 5.1a.
CHO-K1 cells exposed to 1.0-10.0 mM DMPT for 24 hours, there was a mild increase of CMF value. Comparing to control group (DMSO), it only showed significant CMF value of 139.19% on 7.5 mM DMPT. However, there was a declination of CMF value to 103.65% while treating with 10.0mM DMPT (Fig. 5.2a).
DMAEMA
In low concentrations of DMAEMA, it had mild effect the same as DMPT on ROS content of CHO-K1 cells. However, there was significant difference of DCF present value, it showed an increase at 3.5 mM DMAEMA (117.6%, p < 0.05) in Figure 5.1b.
Interestingly, when CHO-K1 cells exposed to 2.5 mM DMAEMA, DCF has a significant declination (74.3%, p < 0.05).
CHO-K1 cells exposed to DMAEMA showed the results of CMF assays as well as DMPT group. Similarly, there was a slight elevation of CMF value when CHO-K1 cells exposed to DMAEMA. Till CHO-K1 cells exposed to 2.5 mM DMAEMA, a significant rising (135.56%) was noted. But, there was a declination on the highest concentration DMAEMA the same as the result of DMPT group (Fig. 5.2b).
DMABEE
24 hours exposure to 0.1 to 0.5 mM of DMABEE did not change the ROS level obviously. However, while the cells were treated with high concentration of DMABEE (0.75 mM and 1.0 mM), the ROS level elevated apparently. Comparing to negative control group, the ROS level obviously increased around 50% at 0.75 mM and 1.0 mM DMABEE (Figure5.1c).
There was a dramatic increase of CMF value while CHO-K1 cells exposed to DMABEE. First, while treating with 0.1 mM DMABEE, the percentage of CMF value (211.19%) showed about 2 times of control group. Further, when CHO-K1 cells were incubated with higher concentration DMABEE (0.5, 0.75, 1.0 mM), the CMF value kept around 250% (Fig. 5.2c).
4.1.6 CBMN assay with IF stain DMPT
The results of CBMN assay with immunofluorent staining has been reported to be correlated with measuring DNA damage and cytotoxicity. Thus, in the study, the occurrence of DNA damage by different chemicals was demonstrated by CBMN assay.
A 24 hour exposure of CHO-K1 cells to DMPT indicated a slight increase in ratio of the cells with mononucleus (from 19.8% to 27.1%). On the other hand, the ratio of binucleated cells decreased gradually (from 85.5% to 67.6%) (Fig 6.1a). However, the variation was not dependent on the concentration of DMPT, there was only a significant change at the highest concentration. The percentage of MNi increased form 2.9% to 8.3% in a dose-related pattern (Fig 6.1b).
DMAEMA
The ratio of CHO-K1 cells with mononucleus increased slightly without dose- dependent manner (form 8.93% to 20.0%), however, the ratio of the binucleated cells showed a contrary results (from 88.5% to 76.6%) (Fig 6.2a). Although DMAEMA could affect mitosis of CHO-K1 cells, DMAEMA only mildly brought up the percentage of MNi. It showed variation only in the highest concentration of DMAEMA (5.0%) (Fig 6.2b).
DMABEE
After exposure to DMABEE 24 hours and staining with DAPI and rhodamine phalloidin, the ratio of the mononuclear cells increased dramatically in a
dose-dependent manner (from 8.6% to 34.6%). In contrary, the binuclear cells showed an opposite performance (from 89.1% to 64.3%) (Fig 6.3a). As well as the ratio of the mononuclear cells, the percentage of MNi showed a notable rising in a dose-dependent manner. It demonstrated the increasing trend from 2.7% to 7.2%. Furthermore, there was a declination of the amount of MNi cells exposed to 7.5 mM (Fig 6.3b). In the highest concentration of DMABEE (1.0 mM) in the study, however, the viable cell sharply decreased and the cellular morphology got incomplete. It was too hard to observe the DNA damage and measure the nuclear variation. Thus, we did not show the document of CBMN assay of CHO-K1 cells exposed to 10.0 mM DMABEE.
4.2 Effects of NAC, catalase, & carboxylesterase on DMPT-, DMAEMA- or DMABEE-induced cell growth inhibition.
4.2.1 Effect of NAC, catalase and carboxylesterase on CHO-K1 cells NAC
Theoretically, NAC could decrease the toxic impact that ROS induced on the inhibition of cell growth and proliferation in CHO-K1 cells. After pre-treating with NAC for an hours, CHO-K1 cells exposed to DMPT and DMABEE, however, did not show significant improvement in cell growth inhibition (Fig 7.1a & b). Comparing to control group, there were declinations about 46.75% and 39.51%, respectively, in 1mM and 5mM NAC co-incubation with 10.0 mM DMPT. Moreover, CHO-K1 cells exposed to 1.0 mM DMABEE after pre-treating with 1.0mM / 5.0mM NAC, there were dramatic
declinations in MTT assays. Comparing to control group (1.0mM DMABEE only), the proliferation rate of CHO-K1 cells decreased about 30% to 38%.
The results did not show rehabilitation of the cell growth ability, after incubating with either low or high concentration NAC.
Catalase
In this study, the growth ability would slightly rise while pre-treating with catalase for an hours before incubation with DMPT 10.0 mM or DMABEE 1.0 mM. Treated with DMPT 10.0 mM or DMABEE 1.0 mM, the growth ability of CHO-K1 cells significantly reduced without a concentration-relate manner. While co-treating with various concentration of catalase (500 U or 1000 U), there was a mild increase of proliferation rate about 10% in DMPT and 20% in DMABEE group (Fig 7.2a & b).
Carboxylesterase
When pre-treatment with esterase 1 U/ml or 5 U/ml for an hour and co-incubation with two chemicals (DMPT 10.0 mM and DMABEE 1.0 mM) for 24 hours, the grwth inhibition of CHO-K1 cell might be prevented (Fig 4.3a & c). Especially, the cytoxicity caused by DMABEE could be inhibited obviously, the MTT assays showed that the proliferation rate almost rehabilitated to 94% (Fig 4.3c). However, CHO-K1 cells exposed to DMAEMA co-treating with carboxylesterase did not show significant change (Fig 7.3b).
In morphology, CHO-K1 cells could return to original cuboid or round shape after treating with carboxylesterase.
Chapter V. Discussion
5.1 Morphological and proliferation aberrations
Numerous investigations have revealed that chemicals at milli-molar concentration level eluted from dental restorative material could be found in human tissues or organs (Cetinguc, Olmez et al. 2007). Such concentration are high enough to induce cytotoxicity or genotoxicity. These materials used in dentistry can have harmful effects, manifested mainly as pulp damage or allergic reactions (Schwengberg, Bohlen et al.
2005). In spite of Bis-GMA, TEDGMA and HEMA which are mostly investigated, there are still some initiators / co-initiators, reducing agent and accelerators that should be noted on their toxicity after resins are cured.
In our study, DMPT, DMAEMA and DMABEE were capable of reducing CHO-K1 proliferation rate. Growth assays provided the first evidence that evaluated concentrations of the three substances have chemical and biological effects on CHO-K1 cells. Increasing concentration of DMPT lead to a mild reduction, there was only a 22%
declination in cell numbers (p<0.05) in a short-term exposure of 24 hours. When CHO-K1 cells were exposed to DMAEMA, the growth assay showed the same results as DMPT. Furthermore, the effective inhibitory concentration (about half cell numbers decreasing) was 1.0 mM for DMABEE (p<0.05). Only the results of the growth assays of the cells exposed to DMABEE concurred with the published data. In the previous study, the effective inhibitory concentration demonstrated by Permanent 3T3 Cells, Primary human gingival fibroblasts (HGF), Primary human pulp fibroblasts (HPF), and Primary human periodontal ligament fibroblasts (HPLF) was around 1.22 to 1.26 mM in
treating with DMABEE (Geurtsen, Lehmann et al. 1998). Other two chemicals indicated less cytotoxicity than the results of previous studies. However, the long-term
treating with DMABEE (Geurtsen, Lehmann et al. 1998). Other two chemicals indicated less cytotoxicity than the results of previous studies. However, the long-term