GSH depletion

GSH redox status is vital for biological homeostasis maintenance. CMF fluorescence has been widely used to measure the intracellular levels of reduced GSH. In the experiment, GSH depletion occurred at cells treated with each one of three chemicals.

Interestingly, CMF value of CHO-K1 cells treated to both DMPT and DMAEMA, it showed a similar results. The mean GSH value achieved the highest, which was about 1.5 times of control cells at 7.5 mM DMPT or 2.5 mM DMAEMA.The reduced GSH content might induce and indicate apoptosis. The cells that could quickly generate more GSH adapt the toxicant challenge and maintain viability. This might also be explained by the heterogeneity of cells (Chang, Ho et al. 2001). However, there was a slight GSH depletion at the highest concentration of DMPT or DMAEMA, it implied that CHO-K1

cells would start to apoptosis while incubating with high concentration DMPT or DMAEMA.

Intracellular GSH content of individual cell might be crucial for its survival. Thus, if cells contacted with more toxic materials, the GSH level would increase more. As CHO-K1 cells were treated with DMABEE, which was the most cytotoxic drug in this study, GSH level prominently increased. This might indicate that in order to survival, the residual CHO-K1 cells produced more GSH to against the environmental stress.

However, all of this three substances did not induce notable GSH depletion by CMF flow cytometry assay. Possibly ROS accumulation induced by the three chemicals is not be secondary to GSH depletion. Other mechanism should be considered.

5.6 DNA damage

Micronuclei, which mean DNA breakdown, originate from chromosome fragments or whole chromosomes that lag behind at anaphase during nuclear division. CBMN assay is the preferred method for measuring MNi in cultured human or mammalian cells because scoring is specifically restricted to once-divided binuclear cells, which are the cells that can express MNi (Fenech and Morley 1986). Thus, in our study, we used CBMN assay as a quantitative scoring assessment of DNA damage.

A source of free radicals is required for the initiation of the polymerization process of dental resins. Pagoria et al. showed that these initiating radicals indiscriminately react with molecular oxygen forming ROS, which may damage DNA (Pagoria, Lee et al.

2005). We wanted to analyze the severity of oxidative DNA damage elicited by different chemicals, such as DMPT, DMAEMA and DMABEE.

In our study, the negative control, CHO-K1 cells incubated with DMSO in F-12 medium for 24 hours, contained an average around 2.6% MNi production. On the contrary, the positive control, CHO-K1 cells incubated with mitomycin C in F-12 medium for 24 hours, showed an average around 11.6% MNi production. After incubating with DMPT, the percentage of MNi in BN cells increased in a dose-dependent manner (Fig. 3.1b). The result implied that chromosome of CHO-K1 cell might be attacked by DMPT or the metabolites of DMPT and this kind of DNA damage could not be repaired. DMAEMA is a co-initiator as well as DMPT. However, it showed minor genotoxicity and the MNi ratio increased slightly only at higher concentrations (3.5 mM DMAEMA) comparing to the positive control. Both the results of treating with DMPT and DMAEMA were coincident to the research of Nomura et al. The genotoxicities of DMPT was thought to be moderate and DMAEMA did not appear to be genotoxic (Nomura, Teshima et al. 2006). Among three chemicals used in this study, DMABEE was the most genotoxic and would induce cellular breakdown. An obvious elevation of MNi while incubating CHO-K1 cells with DMABEE were detected. But, treating with 0.75 mM DMABEE, there was a dramatic declination of the percentage of MNi. The reason might be because cells were morphologically altered at the high concentration of DMABEE as well as the figures shown in MTT assays (Fig. 2.3a & b).

The cellular image observed under IF staining also displayed a broken cell of skeleton and incomplete nucleus. Moreover, as CHO-K1 treated with 1.0 mM DMABEE, cells were severely disrupted and could not be scored for calculation.

5.7 Inhibitors & enzymes

NAC was regarded as an inhibitor that alleviated genotoxicity and cell cycle arrest induced by monomers, such as TEGDMA and HEMA, and down-regulated ROS level caused by HEMA (Schweikl, Hartmann et al. 2007). Whereas, in our study, either NAC did not decrease growth inhibition and ROS production induced by DMPT and DMABEE. But interestingly, the ROS scavengers NAC could enhance growth inhibition while co-incubating with DMABEE (Fig. 4.1b). This observation supplements data from a previous study. It was found that NAC may obviously behave as antioxidant or pro-oxidant either alone or in combination with vitamin C dependent on the applied concentration. Thus, NAC, about 0.5 to 1.0 mM, produced significantly more DNA damage compared to the exposure of DNA to CQ / DMPT without NAC (Lee, Pagoria et al. 2007).

Catalase is the main element in antioxidant defense system to decompose hydrogen peroxide. However, CHO-K1 cells pre-treated with catalase did not enhance growth ability compared to the cells treated with DMPT or DMABEE alone. The results implied that hydrogen peroxide could not be one of the ROS product induced by DMPT or DMABEE.

In enzymology, the CES are a group of enzymes that catalyze the chemical reaction of hydrolysis of ester- and amide-bond-containing substances. The two substrates of this enzyme are carboxylic ester and water, whereas its two products are alcohol and carboxylate. Saliva contains various types of esterase are from this group belong to the family of hydrolases and act on carboxylic ester bonds. CES reduces the toxicity induced by chemicals with ester, amide or carbamate groups. In our study, the growth