Study On The Oxidative Damage And Alternation Of γ-GCS Expression In Hepatocytes Of Mice Exposed To Microcystin-LR

Objective To study the oxidative damage and alteration of mRNA andprotein expression of γ-glutamylcysteine synthethase (γ-GCS) subunit inhepatocytes of mice exposed to microcystin-LR（MC-LR），and explore therelationship between oxidative damage and expression of γ-GCS in mousehepatocytes.Methods Eighty mice aged5weeks were divided randomly into controlgroup which was administered with0.005ml/g of saline contained0.02%dimethyl sulfoxide,5,10and20μg/kg groups. MC-LR was administered for10and20days respectively by intraperitoneal injection once a day. The mice wereweighed every two days. By the end, the mice were routinely sacrificed afterexposure to MC-LR for10and20days respectively, and the livers were quicklyremoved. The levels of malondialdehyde and glutathione in the liver weredetermined by thiobarbituric acid （TBA） and dithio-bis-nitrobenzoic acid（DTNB）methods respectively. Immunohistochemistry was applied to observethe level of8-hydroxydeoxiguanosin which is the marker of DNA oxidativedamage. In addition, total RNA were extracted from the liver and reverse transcribed into cDNA. Quantitative real-time PCR was performed to determinethe expression of GCLC and GCLM mRNA. Protein in the mice livers wasextracted and expression of GCLC and GCLM protein were measuered bywestern blot.Result1. Growth rate of body mass of the mice in10μg/kg and20μg/kggroup were significantly decreased compared with control group exposed toMC-LR for10and20days（P<0.05）, and were decreased in a dose-dependentmanner.2. The level of MDA in the mice liver in10μg/kg and20μg/kg groups wereelevated compared with control group exposed to MC-LR for10and20dayswhich were statistically significant（P<0.05）.3. The level of GSH in the mice liver in10μg/kg and20μg/kg groups weresignificantly higher than control group after10days exposure（P<0.05）. Thelevel of GSH in MC-LR treated groups were significantly decreased comparedto control group（P<0.05）after exposure for20days.4. The level of8-OHdG in MC-LR treated groups were significantlyincreased compared to control group（sP<0.05）after exposure for10and20days,and which were increased in a dose-dependent manner.5. The expression of GCLC mRNA in the mice liver was down regulated inMC-LR treated groups after exposure for10days，which were respectivelyreduced to72.0%,44.1%and46.5%of those in control group. In addition theexpression of GCLC mRNA in MC-LR treated groups were respectivelyreduced to68.5%,26.6%and26.7%of those in control group after20daysexposure（P<0.05）.6. The expression of GCLM mRNA in MC-LR treated groups were lowerthan control group after10days exposure, which fell to59.4%,42.5%and 34.3%of those in control group（P<0.05）. Moreover, the expression of GCLMmRNA were a downward trend in MC-LR treated groups, which were reducedto62.9%,31.9%and47.7%of those in control group after20days exposure（P<0.05）.7. The expression of GCLC protein in the mice liver in5μg/kg group wasno significant alteration exposed to MC-LR for10days（P>0.05）, but which in10μg/kg and20μg/kg groups were significantly decreased compared withcontrol group（P<0.05）. Besides the expression of GCLC protein in MC-LRtreated groups were lower than control group, which were statistics significantlyafter20days exposure（P<0.05）. Furthermore, the expression of GCLC proteinin MC-LR treated groups exposed for20days were lower than of those in10days exposure groups (P<0.05).8. The expression of GCLM protein in the mice liver in5μg/kg group hadno significant difference compared with control group exposed for10days（P>0.05）, but which in10μg/kg and20μg/kg groups were lower than those incontrol group （P<0.05）. Morever, the expression of GCLM protein inexperiment groups were significantly lower than control group after20daysexposure (P<0.05). In addition, the expression of GCLC protein in MC-LRtreated groups exposed for20days were lower than of those in10days exposuregroups（P<0.05）.9. The expression of GCLC and GCLM mRNA were positively correlationwith the levels of MDA（rGCLC=0.425, P<0.05, rGCLM=0.304，P<0.05）in the micehepatocytes. Morever, the expression of GCLC and GCLM mRNA were alsopositively correlated with the level of8-OHdG （rGCLC=0.420, P<0.05；rGCLM=0.476, P<0.05）.Conclusion MC-LR induced the down-regulation of γ-GCS mRNA and protein expression as well as a decrease in GSH levels in mouse hepatocytes. Inaddition, MC-LR significantly increased the levels of MDA and8-OHdG, andinduced the oxidative damages in lipid and DNA. Further studies are required toclarify the mechanisms of down-regulation of γ-GCS by MC-LR.