The protective role of selenium against dental amalgam-induced intracellular oxidative toxicity through the TRPV1 channel in DBTRG glioblastoma cells

Abstract Objective The exposure to mercury (Hg) from dental amalgams is a suspected causative factor in neurological diseases. This study investigated the toxic effects of two different amalgam compositions related to Hg and the protective effects of selenium against the toxic effects of Hg through the TRPV1 channel in the human DBTRG glioblastoma cell line. Methodology Six groups of the cells were organized. Analyses of cell viability, apoptosis, caspase 3 and caspase 9 activities, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, and Western Blotting for protein expression levels were performed. Results Cell viability values were lower in amalgam with high copper (HCu) and low copper (LCu) groups independently of time but were increased by selenium and capsazepine (p<0.001 and p<0.05). Conversely, apoptosis rates, caspase 3 and caspase 9 expression, ROS formation, mitochondrial membrane depolarization, and protein expression levels were higher in the HCu and LCu groups but were decreased by selenium (p<0.001 and p<0.05). Conclusions Selenium combined with an amalgam of either HCu or LCu decreases the toxic effects created by Hg in human DBTRG glioblastoma cells.


Introduction
Dental amalgam contains approximately 50% elemental mercury (Hg 0 ), the exposure to which from dental amalgams is a suspected causative factor in neurodevelopmental and neurodegenerative diseases. 1 Accordingly, developing new approaches to clinical applications is essential to prevent exposure to the possible negative effects of Hg in dental amalgams.
The toxic effects of dental amalgam, which has been in use in dentistry for more than 150 years, and the possible side effects of Hg from dental amalgams have been the subject of several studies. Amalgam Hg has a selenophilic property and shows a higher affinity for Se than for thiol groups. 1 Previous studies investigating Se effect have reported a reduction in the cytotoxicity of amalgam, whereas others suggest otherwise. 13,14 Selenoprotein P -a Se transport protein -is an important extracellular antioxidant that is essential for neuronal survival and function. 15 obtained from these sources: Dihydrorhodamine-123 (DHR123) and tris-glycine gel were from Molecular Probes (Eugene, OR, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase 3 substrate (AC-DEVD-AMC) were from Sigma-Aldrich (Madrid, Spain), and caspase 9 substrate (AC-LEHD-AMC) was from Bachem (Bubendorf, Switzerland); 5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-

Preparation of amalgams
The pre-dosed amalgam capsules were mixed using a high-energy mixer (Silamat S5, Ivoclar Vivadent AG, Liechtenstein) for 5-7 seconds, in accordance with the manufacturers' instructions, and then transported to the cell culture media according to the groups. 19 The

Groups
The cells in all groups were counted using an automatic cell counter to achieve 1×10 6 cells per flask, which were divided into six main groups (Table 1). 17,20 For the analyses, the cells were further treated with CAP (0.01 mM, channel-specific agonist) to activate the TRPV1 channel, and, when necessary, were inhibited with the TRPV1 channel blocker CpZ (0.1 mM, channel-specific antagonist). 21 CAP and CpZ were dissolved in DMSO for the preparation of the stock solution, with phosphate-buffered saline (PBS) used for dilution, and the pH was adjusted. The amalgams were added directly to the cell culture medium. The stock solution was prepared in sterile distilled water for Se (0.2 mM) and diluted (10 6 times) to achieve the final concentration. After incubation, the control and treated cells were examined for cell viability, caspase 3 and caspase 9, apoptosis, intracellular ROS production and mitochondrial membrane depolarization, and for Western Blotting analyses.
The assay uses a dye that is selectively imported by cells undergoing apoptosis, and, since necrotic cells cannot retain the dye, they are not stained. The accepted statistical significance limit was p<0.05.

Intracellular ROS and JC-1 values
The intracellular ROS and JC-1 values in the six groups at 2, 12, 24 and 48 h are presented in Figures   1, 2, 3 and 4, respectively. The intracellular ROS levels of the LCu and HCu groups were significantly higher than in the control and Se groups (p<0.001 and p<0.05). In the six groups, a decrease over time     in amalgams have been thought to enhance their cytotoxic effects, low-Cu and high-Cu amalgams have been shown to have the same cytotoxicity level. 14 Cu ratio effect on amalgam toxicity was also considered in our study, with amalgams with different Cu ratios chosen for study; however, no difference between the groups was found, in consistence with previous studies. Secondary changes in DNA due to the attachment of Hg to thiol groups may be important.

Conclusion
We found that Se has an anti-apoptotic effect on human DBTRG glioblastoma cells, decreasing Ca 2+ influx, mitochondrial oxidative stress and apoptosis by the modulation of the cell TRPV1 channel activity.
Se may thus be useful as an anti-apoptotic agent, since it decreases Hg toxic effects in human DBTRG glioblastoma cells when combined with an amalgam of either HCu or LCu. The production of amalgams with Se content can prevent toxicity by limiting Hg release, although further laboratory studies are needed to produce such materials.