Effects Of Oxidative Stress Induced By Microcystin LR On Repair Genes Of Rat Hepatocytes Brl

Objectives:To determine the effects of microcystin-LR on cellular proliferation, apoptosis, oxidative injury and the expression changes of repair gene JWA, XRCC1 and PRAP1 by in vitro culture of rat hepatocytes BRL, and further to explore the effects of oxidative stress induced by microcystin LR on repair genes of rat hepatocytes BRL.Methods:1.Different doses (0, 1, 5, 10, 30μg/ml) of microcystin-LR were used to exposure the rat hepatocytes BRL-3A for 24h, 48h and 72h, respectively. MTT assay was used to determine the cellular proliferation and apoptosis.2. Different doses (0, 1, 5, 10,30μg/ml) of microcystin-LR were used to exposure the rat hepatocytes BRL-3A for 48h, than the cellular glutathione peroxidase (GSH-PX), superoxide dismutase (T-SOD), malondialdehyde (MDA) content and the leakage rate of lactate dehydrogenase (LDH) were determined .3. Different doses (0, 1, 5, 10, 30μg/ml) of microcystin-LR were used to exposure the rat hepatocytes BRL-3A for 24h, 48h and 72h, respectively. Real-time fluorescence quantitative PCR was used to determine the expression alteration of JWA, XRCC1 and PARP1 genes.Results:1. Compared with the control , different doses (1, 5, 10, 30μg/ml) of microcystin-LR could inhibit the growth of rat hepatocytes BRL-3A. The result shows that the cell number was decreased in a dose-response relationship and time-effect relationship.2. Compared with the control , the amounts of intracellular MDA increased with which different doses (1, 5, 10, 30μg/ml) of microcystin-LR exposured for 48h , but it had no statistical difference; and decreases of superoxide dismutase ( T-SOD) and glutathione peroxidase (GSH-PX) levels were found in the dose of 30μg/ml microcystin-LR.Treatment with 10 and 30μg/ml microcystin-LR for 48h the intracellular leakage rate of lactate dehydrogenase (LDH) was increased , and its difference was statistically significant.3. Compared with the control group , the gene expression of PARP1 was decreased in the group which the 30μg/ml microcystin-LR was used to exposure the rat hepatocyte BRL-3A for 48h. The gene expression of JWA, XRCC1 and PARP1 were decreased in the group at the same condition for 72h.Conclusions:1.The doses of 1-30μg/ml microcystin-LR could inhibit the growth of rat hepatocyte BRL-3A in a dose-response relationship and time-effect relationship.2. The microcystin-LR could induce oxidative stress, and the leakage rate of lactate dehydrogenase (LDH) was increased with treated by 10 and 30μg/ml microcystin-LR. Under 30μg/ml microcystin-LR treatment, the intracellular superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) decreased.3. In our experimental conditions, microcystin-LR could induce the alteration of repair gene of JWA, XRCC1 , PARP1 and apoptotic genes of bax, P53 in rat hepatocyte BRL-3A. Exposured to 30μg/ml microcystin-LR for 48h, the gene expression of PARP1 was decreased, and after 72h exposure, the gene expression of JWA,XRCC1 and PARP1 all were decreased.