Conclusion - 三種牙科樹脂之化學成分毒性效應之分析

Resin-base products are widely used in modern dentistry. Chemical substances leached from the materials may potentially cause adverse effects. In order to improve the safety problems, the toxic mechanism and cytoxicity of these chemicals should be investigated. It is reliable to check the mechanism through examination of cell cycle progression, ROS production and GSH alteration. Here we tested three chemicals of composite resins: DMPT, DMAEMA and DMABEE via these aspects, hope to find out the implication for improvement of their clinical security.

DMPT induced both short-term and long-term growth inhibition in CHO-K1 cells in a dose-dependent manners. However, there were not obvious cell cycle deregulation, ROS overproduction / GSH depletion and the cellular distribution was not change significantly by Annexin V-FITC / PI. When the cells were exposed to DMPT, the ratio of MNi was increased. Only through DNA damage could explain the source of cytotoxicity.

DMAEMA produced growth inhibition of CHO-K1 cells in a dose-related manner, especially in long-term growth inhibition. Amount three chemicals, the cytotoxicity of DMAEMA was weaker. Whether ROS production / GSH depletion or DNA damage, there were no obvious results that could explain of cytotoxicity. However, a sub-lethal dose of DMAEMA induced cell cycle disturbance in S phase and G0/G1phase.

Therefore, we should find out other mechanisms to explain the toxicity of DMAEMA.

DMABEE elicited growth inhibition of CHO-K1 cells in dose-dependent manner, which may be related to cell cycle perturbation, ROS overproduction and DNA damage.

CES and catalase may attenuate toxicity. On the other hand, NAC, as well as a

pro-oxidant, exacerbated cytotoxicity of DMABEE. DMABEE induced cell cycle arrest in G0/G1 phase which could increase the percentage of apoptotic cells evaluated by Annexin V-FITC / PI assay.

The results of this study help to elucidate the toxicity of these chemicals. Many in vitro studies indicate that the dosage of the leachable substances may not reach the toxic level as we reported. However, in some clinical situations, the unbound chemicals may lead to potential toxic effects as we addressed. Thus, for the safety of clinical application, it is important to realize the toxic mechanism of these substances and down-regulate the cytotoxicity as well as possible.

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(a)

(b)

Fig. 1.1a & b Effect of DMPT on the growth of CHO-K1 cells.

(a) Cells (1.0 × 10⁵ cells) in 6-well plate were exposed to DMPT for 24 hours and were measured by MTT assay. (b) Cells (200 cells) in 6-well plate were exposed to DMPT, then were cultured for 7 days and calculated by CFC assay. Results were expressed as percentage of control (mean ± SE).

*Denotes marked difference when compared with control.

(n=6)

(n=4)

(a)

(b)

Fig. 1.2a & b Effect of DMAEMA on the growth of CHO-K1 cells.

(a) Cells (1.0 × 10⁵ cells) in 6-well plate were exposed to DMAEMA for 24 hours and were measured by MTT assay. (b) Cells (200 cells) in 6-well plate were exposed to DMAEMA, then were cultured for 7 days and were calculated by CFC assay. Results were expressed as percentage of control (mean ± SE). *Denotes marked difference when compared with control.

(n=6)

(n=4)

(a)

(b)

Fig. 1.3a & b Effect of DMABEE on the growth of CHO-K1 cells.

(a) Cells (1.0 × 10⁵ cells) in 6-well plate were exposed to DMABEE for 24 hours and were measured by MTT assay. (b) Cells (200 cells) in 6-well plate were exposed to DMABEE, then were cultured for 7 days and were calculated by CFC assay. Results were expressed as percentage of control (mean ± SE). *Denotes marked difference when compared with control.

(n=6)

(n=4)

DMPT 10.0 mM DMSO (NC)

(a) (b)

DMAEMA 3.5 mM DMSO (NC)

Fig. 2.2a & b Morphology alternation of CHO-K1 cells.

(a) Control CHO-K1 cells. (b) CHO-K1 exposed to 3.5 mM. DMAEMA for 24 hours. (50x, original magnification)

(a) (b)

Fig. 2.1a & b Morphology alternation of CHO-K1 cells.

(a) Control CHO-K1 cells. (b) CHO-K1 exposed to 10.0 mM. DMPT for 24 hours. (100x, original magnification)

(a) (b)

DMSO (NC) DMABEE 1.0 mM

Fig. 2.3a & b Morphology alternation of CHO-K1 cells.

(a) Control CHO-K1 cells. (b) CHO-K1 exposed to 1.0 mM. DMABEE for 24 hours. (100x, original magnification)

DMPT 10.0 mM DMPT 10.0 mM

Fig. 2.4a & b Morphology alternation of CHO-K1 cells via immunofluorescence staining.

(a) Nucleus of CHO-K1 cells stained with DAPI (blue), and (b) actin stained with rhodamine phalloidium. (100x, original magnification)

57 Induction of necrosis and apoptosis of CHO-K1 cells by DMABEE

Dot gram analysis by flow cytometry of CHO-K1 cells treated with DMSO (as NC) and DMABEE (0.1mM-1.0 mM). Result of one representative experiment from each group was shown.

(b)

(c)

Fig. 3.1a, b & c Effect of three chemicals analysis by Annexin V-FITC / PI assay flow cytometry.

Cells (2.5 × 10⁵ cells) in 6-well plate. (a) Cells were exposed to DMPT for 24 hours and were measured by Annexin V-FITC / PI flow cytometry. (b) Cells were exposed to DMAEMA for 24 hours and were measured by Annexin V-FITC / PI flow cytometry. (c) Cells were exposed to DMABEE for 24 hours and were measured by Annexin V-FITC / PI flow cytometry.

(n=7) (n=8)

(n=5)

Effects of DMSO on the cell cycle progression (G0/G1; S; G2/M phase) of CHO-K1 cells

Histogram analysis by flow cytometry of CHO-K1 cells treated with DMSO. Result of one representative experiment from control group.

Marker % Gated

0 200200 400400 600600 800800 10001000 FL2-A

Effects of DMSO on the cell cycle progression (Sub-G0/G1 phase) of CHO-K1 cells

Histogram analysis by flow cytometry of CHO-K1 cells treated with DMSO. Result of one representative experiment from control group.

Fig. 4.1a, b & c Effect of three chemicals on the cell cycle of CHO-K1 cells (G0/G1, S, G2/M).

Cells (2.5 × 10⁵ cells) in 6-well plate. (a) Cells were exposed to DMPT for 24 hours and were measured by PI flow cytometry. (b) Cells were exposed to DMAEMA for 24 hours and were measured by PI flow cytometry. (c) Cells were exposed to DMABEE for 24 hours and were measured by PI flow cytometry.

(a) (b)

(c)

CHOK1-250000cells-DMPT dose-24H-PI test(G0/G1;G2/M;S)-24h

(n=6)

CHOK1-250000cells-DMABEE dose-24H-PI test(G0/G1;G2/M;S)-24h

(n=6)

(n=4)

Fig. 4.2a, b & c Effect of three chemicals on the cell cycle of CHO-K1 cells (Sub-G0/G1).

(a) (b)

(c)

(n=6) (n=6)

CHOK1-250000cells-DMABEE dose-24H-PI test(Sub-G0/G1)-24h (n=4)

0 200200 400400 600600 800800 10001000 FSC-H

Effects of DMPT on the DCF expression of CHO-K1 cells

Histogram of DCF fluorescence of CHO-K1 cells treated with 0 to 10.0 mM DMPT. M2 population indicated ROS content were noted. Result of one representative experiment was shown

(b)

Fig. 5.1a, b & c Effect of three chemicals analysis by DCF flow cytometry

Cells (2.5 × 10⁵ cells) in 6-well plate. (a) Cells were exposed to DMPT for 24 hours and were measured by DCF flow cytometry. (b) Cells were exposed to DMAEMA for 24 hours and were measured by DCF flow cytometry. (c) Cells were exposed to DMABEE for 24 hours and were measured by DCF flow cytometry.

(n=6) (n=6)

(n=6)

DMABEE

0 200200 400400 600600 800800 10001000 FSC-H

Effects of DMPT on the CMF expression of CHO-K1 cells

Histogram of DCF fluorescence of CHO-K1 cells treated with 0 to 10.0 mM DMPT. M2 population indicated GSH depletion content were noted. Result of one representative experiment was shown

(n=4)

(b)

Fig. 5.2a, b & c Effect of three chemicals analysis by CMF flow cytometry

Cells (2.5 × 10⁵ cells) in 6-well plate. (a) Cells were exposed to DMPT for 24 hours and were measured by CMF flow cytometry. (b) Cells were exposed to DMAEMA for 24 hours and were measured by CMF flow cytometry. (c) Cells were exposed to DMABEE for 24 hours and were measured by CMF flow cytometry.

(n=4) (n=4)

(n=4)

CMF CMF

CMF

(a) (a)

(b)

Fig. 6.1a & b CBMN assay of CHO-K1 cells.

Cells (1.0 × 10⁵ cells) in 6-well plate on 18 × 18 mm slides were exposed to DMPT for 24 hours and the group of mitomycin c was regarded as positive control. (a) The ratio of mononuclear and binuclear cells were calculated by CBMN assay with IF staining. (b) MN / binuclear cells ratio was measure by CBMN assay (the number of binuclear cells > 500, at least)

(n=4)

(a) (a)

(b)

Fig. 6.2a & b CBMN assay of CHO-K1 cells.

Cells (1.0 × 10⁵ cells) in 6-well plate on 18 × 18 mm slides were exposed to DMAEMA for 24 hours and the group of mitomycin c was regarded as positive control. (a) The ratio of mononuclear and binuclear cells were calculated by CBMN assay with IF staining. (b) MN / binuclear cells ratio was measure by CBMN assay (the number of binuclear cells > 500, at least)

(n=4)

(a) (a)

(b)

Fig. 6.3a & b CBMN assay of CHO-K1 cells.

Cells (1.0 × 10⁵ cells) in 6-well plate on 18 × 18 mm slides were exposed to DMABEE for 24 hours and the group of mitomycin c was regarded as positive control. (a) The ratio of mononuclear and binuclear cells were calculated by CBMN assay with IF staining. (b) MN / binuclear cells ratio was measure by CBMN assay (the number of binuclear cells > 500, at least)

(n=4)

Fig. 7.1a & b Effect of CHO-K1 cells co-treating with NAC on the growth ability.

Cells (1.0 × 10⁵ cells) in 6-well plate were incubated with NAC for an hour. (a) Cells were exposed to DMPT for 24 hours and were measured by MTT assay. (b) Cells were exposed to DMABEE for 24 hours and were measured by MTT assay.

(b) (a)

(n=4)

* :p<0.05 compare with control