Phellinus Linteus Mushroom Protects Against Tacrine-induced Genotoxicity In HepG2 Cells

Tacrine was the first drug approved for the treatment of Alzheimer's disease by FDA and was thought to exert its therapeutic effects via inhibition of acetylcholinesterase.However,reversible hepatotoxicity limits its clinical use.The patients with Alzheimer's disease who received treatment of tacrine for 2 to 4 weeks would be found with serum alanine aminotransferase levels increasing.Sometimes the levels could increase 3 times even 20 times in a small percent and some cases with jaundice.The mechanism of tacrine-induced hepatotoxicity is not clear.Many in vitro studies suggest that oxidative stress is a main mechanism involved in tacrine-induced hepatotoxic cytotoxicity.In our previous study,we found that tacrine usually accumulated in mitochondria,inducing generation of excessive ROS in cells,inhibited mtDNA synthesis,cytochrome c release,caspase-3 induction and subsequently hepatocyte apoptosis.It has been reported that the efficacy of many drugs derived from genotoxicity mechanism and also make damage to normal cells of human.Drugs which used for clinical chemotherapy for example cisplatin,carboplatin and Adriamycin are used to kill tumor cells by mechanism of ROS-induced DNA damage and prevent cell division,but the division of normal cells will be also affected in the same mechanism,thus the adverse effect of antitumor therapy was derived from this mechanism.Changes of gene information would be found in normal cells caused by antitumor therapy.The survival of human would be affected by chromosome gene information changing derived from genotoxicity in different degree,from mutation to death of cells.Some study shows that tacrine induced unscheduled DNA synthesis in hepatocytes and suggest that tacrine may be a genotoxic rodent carcinogen.But using different model,the results about genotoxicity of tacrine are different.So,the first object of our present study is to further certify tacrine-induced genotoxicity in the HepG2 cells.The HepG2 cell line was originally derived from a human hepatoblastoma,retaining many functions of normal liver cells and the activities of several phase I and II xenobiotic metabolizing enzymes.Nowadays,HepG2 cells have been confirmed to be a suitable system for determine the genotoxicity,anti-cytotoxicity and cytoprotective effects.Phellinus linteus(PL)is a mushroom that has been widely used as a folk medicine in Asian countries to treat digestive system diseases and gynecological diseases.It has been used as a traditional herbal medicine in China for hundreds of years to cure gastrointestinal disease and was reported to have various pharmacological properties including hepatoprotective,anti-cancer,anti-oxidative,and anti-inflammatory effects in modern medical research.Many studies have also shown that PL had antioxidative and anti-inflammatory effects which could prevent liver damage and offer alternatives to the currently used antidiabetic agents.In our previous study,we found that PL could protect against tacrine-induced oxidative stress,mitochondrial impairment and cytotoxicity in HepG2 cells.The main object of present study is to explore the mechanism of whether PL could protect the genotoxicity induced by tacrine in HepG2 cells.Objective: To further certify the mechanism of tacrine-induced genotoxicity is the main mechanism of cytotoxicity in the HepG2 cells,and to explore the mechanism of whether PL could protect the genotoxicity induced by tacrine in HepG2 cells.Method: HepG2 cells were treated with tacrine under different concentrations of 18.8 and 37.5 ?M.To determine the effects of PL,HepG2 cells were pretreated with PL before tacrine-treatments.Micronucleus assay was designed to observe the protective effect of PL for the genotoxicity induced by tacrine in HepG2 cells.Comet assay was designed to observe the DNA damage induced by tacrine.The 2,7-dichlorofluorescein diacetate(DCFH-DA)method was used to assess the amount of ROS formation.An immunoperoxidase method for detecting and quantitating oxidative damage of 8-OHdG in single cells was used.The intracellular GSH level was monitored by OPT assay.The activities of the antioxidant enzymes superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)were measured spectrophotometrically.Result: Tacrine increased the DNA migration,MN formation,the level of 8-OHdG and ROS in a dose-dependent manner and decreased the level of GSH in a dose-dependent manner,respectively.The activities of the antioxidant enzymes SOD,CAT and GSH-Px were all decreased significantly in tacrine-treated cells.When the cells were pretreated with PL,PL significantly reduced tacrine-induced micronuclei frequencies,DNA fragments,8-OHdG formation,and depletion of GSH in HepG2 cells.The activities of the antioxidant enzymes SOD,CAT and GSH-Px were all increased significantly in PL-pretreated HepG2 cells compared to only tacrine-treated cells.Conclusion: These data suggest that tacrine could induce genotoxicity in HepG2 cells,tacrine-induced generation of excessive ROS is one of the main mechanism of genotoxicity in the HepG2 cells.PL could attenuate the genotoxicity induced by tacrine in HepG2 cells.