Excessive amount of reactive oxigen species in organism leads to a cellular state known as oxidative stress. They stimulates osteoclasts differentiation and inhibit differentiation and activation of osteoblasts, which leads to bone resorption and several bone diseases. Oxidative stress through epigenetic mechanisms: DNA methylation, histone modifications and non-coding RNAs, alters expression of several genes involved in bone turnover. Interaction between oxidative stress and epigenetic mechanisms is still not completely understood. The aim of our work was to study three factors that could have impact on epigenetic mechanism in human osteosarcoma cells: oxidative stress, inhibitor of DNA methylation and antioxidant. Cells were exposed to hydrogen peroxide, 5-azacitidine, combination of hydrogen peroxide and 5-azacitidine and combination of hydrogen peroxide and TEMPOL, for 24 and 72 hours. Using real-time PCR we monitored expression of 10 genes (DNMT3A, MBD1, MYST1, HAT1, HDAC6, HDCA9, SIRT1, SIRT6, OPG in RANKL) after exposure to certain conditions. We did not prove any significant changes in gene expression. But according to trends shown in graphs, we deduce that oxidative stress influence on epigenetic mechanisms. We observed increase expression of MBD1, HAT1, MYST1, SIRT6, SIRT1 in HDAC9 and decrease expression of HDAC6. 5-azacitidine decreases expression of DNMT3A and increases expression of SIRT1, SIRT6 and HDCA6. The protective effect of antioxidant TEMPOL was observed, TEMPOL alleviated influence of oxidative stress on gene expression after 24 hours treatment. We also observed effects of certain conditions on RANK/RANKL/OPG signalling pathway. RANKL expression was downreglated after treatment with hydrogen peroxide, but there was no changes in OPG expression. Decrease of RANKL expresison was detected after treatment with 5-azacitidine.
This was one of the first exsperiments to study the connection between oxidative stress and epigenetic mechanisms. This field is interesting for further investigations, which will help to understand how oxidative stress through epigenetic mechanisms influence on bone homeostasis. This is important for better understanding of patogenesis and development new drug substances in treament of bone diseases.