Changes of dynamical balance of free radicals induced by levodopa in rat glia-containing mesencephalic culture fluid

BACKGROUND ： Parkinson disease is neurodegenerative disorders, characterized by a progressive and selective degeneration of nigrostdatal dopaminergic pathway. Its main clinical symptoms include bradykinesia, dgidity, rest tremor and disturbances in balance. Levodopa （L-DOPA） is the ＂gold standard＂ for the symptomatic treatment of Parkinson disease, but L-DOPA is toxic to dopaminergic neurons and the chronic administration of L-DOPA often causes the side effects of motor such as ＂on-off ＂, etc., and its mechanism still has not been completely clarified. OBJECTIVE ： To observe the changes of the content of glutathione （GSH）, activities of glutathione peroxidase （GSH-Px） and superoxide dismutase （SOD） and content of malondialdehyde （MDA） in gila-containing mesencephalic culture fluid after L-DOPA of different concentrations were added. DESIGN： A comparative observation. SETTINGS ： Department of Neurology, Affiliated Hospital of Taishan Medical University; Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS ： The experiment was carried out in the Basic Research Institute, Taishan Medical University from December 2003 to September 2004. Ten postnatal newborn SD rats （within 2 days） were selected, either male or female. Fetal bovine serum （DMEM/F12） was from Gibco Company; L-DOPA and glial fibrillary acidic protein （GFAP） from Sigma Company. Kits for determination of GSH, GSH-Px, SOD and MDA were purchased from Nanjing Jiancheng Bioengineering Research Institute. METHODS ： Gila-containing mesencephalic culture fluid were placed in 24-well culture plate, and L-DOPA of 50, 100 and 500 μmot/L was added to each group, the blank control group was also set. The glia-containing mesencephalic culture fluid selected at 4, 24, 48 and 72 hours respectively to determine the GSH content with colorimetdc quantitative technique, GSH-Px activity with colorimetdc method, SOD activity with xanthine oxidase method and MDA content with thiol-barbituric acid method. MAIN OUTCOME MEASURES ： GSH and MDA contents, GSH-Px and SOD activities in the glia-containing mesencephalic culture fluid at 4, 24, 48 and 72 hours after L-DOPA of different concentrations were added. RESULTS ： In the glia-containing mesencephalic culture fluid after 100 μmol/L L-DOPA was added for 24, 48 and 72 hours, the GSH contents were lower than those in the blank control group [（174.14±39.28）, （161.55± 40.79）, （144.97±57.59） mg/L; （220.66±32.61）, （221.10±32.98）, （220.43±31.98） mg/L, P 〈 0.05]; The GSH-Px activities were lower than those in the blank control group [（4.03±1.05）, （3.99±1.12）, （3.47±1.00） μmol/L; （5.45±1.14）, （5.69±1.21）, （5.49±1.28） μmol/L, P 〈 0.05]; The SOD activities were also lower than those in the blank control group [（42.02±5.08）, （39.38±5.34）, （38.87±5.75）kNU/L; （51.35±8.87）, （51.78±8.96）, （50.99± 9.09） kNU/L, P 〈 0.05]; Whereas the MDA contents were higher than those in the blank control group [（3.51 ± 1.05）, （3.99±1.03）, （4.45±1.58） μmol/L; （2.09±1.13）, （2.18±1.29）, （2.01±1.05） μmol/L, P〈 0.05]. In the gli- a-containing mesencephalic culture fluid after 100 μmol/L L-DOPA was added for 4, 24, 48 and 72 hours, the GSH contents were （172.27±26.07）, （140.15±61.44）, （137.30±50.87）, （121.09±66.07） mg/L, the GSH-Px activities were （3.89±1.20）, （3.56±1.23）, （3.38±1.18）, （3.01±1.09） μmol/L, the SOD activities were （38.18±6.75）, （35.23±7.85）, （4.59±1.24）, （31.42±7.01） kNU/L, which were all lower than those in the blank control group （P 〈 0.05-0.01 ）; The MDA contents were （3.65±0.86）, （3.87±1.14）, （4.59±1.24）, （4.79±1.32） μmol/L, which were higher than those in the blank control group （P 〈 0.05-0.01）. In the gila-containing mesencephalic culture fluid added by 50 μmol/L L-DOPA, the GSH and MDA contents, GSH-Px and SOD activities at each time point were all close to those in the blank control group （P〉 0.05）. CONCLUSION ： L-DOPA of certain concentration can destroy the dynamical balance of free radicals in glia-containing mesencephalic culture fluid, and accelerate the degeneration of neurons, which are in concentration- and time-dependent manners.