Chromium Ions Induced Cytotoxicity And Oxidative Stress In The MG63 Cell Lines

Free radical biology is a rising knowledge. Reactive oxygen species plays an important role in the pathogenesis of a variety of diseases. The antioxidant can be applied to prevent and cure these diseases. So Free radical biology has very close relations with medicine and becomes an active research field. Reactive oxygen species have been implicated in a variety of degenerative processes, diseases, and syndromes including oral chronic inflammation and pathogenic bone resorption.Non-noble metallic materials have been widely used in dental prosthesis. However, some syndromes are observed in clinic such as the hyperplasia and swollen of the gingiva even localized periodontitis around the prosthesis. One of the crucial factors is the prosthetic materials. These alloys release metallic ions to surrounding oral tissues. Some evidences suggest that exposure to certain metal ions causes cytotoxicity, but the mechanisms remain undefined. Cellular oxidative stress, resulting from the imbalance of cellular free radical generation and antioxidative defense, may be a common pathway in cellular responses to metal ions. Bone remodeling depends on a delicate balance between bone formation and bone resorption, wherein bone-forming osteoblast and bone-resorbing osteoclasts play central roles. The metal ions released from dental alloys probably influence the viability of osteoblasts and result in the loss of bones. Therefore, it was necessary to make osteoblasts as a model for evaluating the response to metal ions. Chromium is a basic element in dental alloys. Previous studies implicated the toxicity of chromium in many diseases and pathogenesis. Cytotoxicity induced by chromium ions was studied by many scholars, but the mechanism is not very clear. The present study was designed to test the hypothesis that oxidative stress mediates chromium-induced cytotoxicity in human osteoblast-like MG63 cells and antioxidant NAC can provide protection for osteoblasts against chromium-induced oxidative stress. We assessed the chromium ions effects on cell viability, cell maturation, intracellular ROS levels, and lipid peroxidation (LPO) level in the presence or absence of NAC. There are three major parts in this research.1 The effect of oxidative stress on the viability of MG63 cellsThe MG63 cells were treated with X/XO and the model of oxidative stress in MG63 cell lines was established successfully. Cellular viability and morphology was evaluated by the MTT assay and phase contrast microscope and then, cellular oxidative stress was evaluated by intracellular reactive oxygen species (ROS) production through the oxidation-sensitive fluorescent probe, DCFH-DA and flow cytometry. The results showed that X/XO induced intracellular ROS production in a dose-and time-dependent manner. The cellular viability was reduced and cellular morphology was damaged, too. Oxypurinol(the inhibitor of XO) reduced the ROS production and cytotoxicity induced by X/XO. These results suggest that oxidative stress damaged the cellular morphology and reduced the cellular viability in MG63 cell lines.2 The cytotoxicity induced by chromium ions in MG63 cellsMG63 cells were exposed in vitro to 5-20μM Cr⁶⁺ and Cr⁺³. Also, in order to evaluate the effects of antioxidant NAC on the cytotoxicity induced by chromium ions in MG63 cells, the cells were pretreated with l-5mM NAC for 30 min and then cells were incubated in 10μM Cr⁶⁺ for 24h or 72h. Cellular survival rates and cell differentiation were evaluated by MTT assay and alkaline phosphatase(ALP) activity assay. The results showed that Cr⁶⁺ reduced cellular survival rate and ALP activity. The cellular morphology and ultrastructures were damaged in MG63 cells. 5-20μM Cr³⁺ has no effects on the MG63 cells. Treatment of MG63 cells with l-5mM NAC afforded dose-dependent reduction to the cytotoxicity induced by Cr⁶⁺.3 Oxidative stress induced by chromium ions in MG63 cellsMG63 cells were exposed to 5-20μM chromium ions. Then, cellular oxidative stress was evaluated by intracellular reactive oxygen species(ROS) production and the degree of lipid peroxidation(LPO). MG63 cell lines were dyed by the oxidation-sensitive fluorescent probe DCFH-DA. The fluorescent intensity was measured with flow cytometric analysis. The levels of MDA were used to indicate intracellular lipid peroxidation. A dose-dependent increased intracellular ROS production was found when cells were exposed to 5-20μM Cr⁶⁺ for 1h. A time-dependent increased ROS production was found when cells were exposed to 10μM Cr⁶⁺ during 0-120min. When MG63 cells were treated with 5-20μM Cr⁶⁺for 24h and 72h, from the MDA assay we found that cellular lipid peroxidation increased in a time-and dose-dependent manner. Pretreatment of MG63 cells with l-5mM NAC for 30min afforded reduction to the cellular oxidative stress induced by Cr⁶⁺. On the contrary, Cr³⁺ had no significantly negative influence in MG63 cells in our study(5-20μM).Based on these studies, our results suggest that oxidative stress might be involved in Cr⁶⁺ induced cytotoxicity in osteoblasts. Antioxidant NAC can play a critical role against Cr⁶⁺-induced cytotoxicity. 5-20μM Cr³⁺ has no significantly cytotoxicity in MG63 cell lines and cellular oxidative stress was not found, too.