Studies On The Role And Mechanism Of Chitosan On The Oxidative Damage Of HepG2 Cell Induced By Cadmium Chloride

Chitosan, 2-amino-2-deoxy-β-D-glucose, with the formula of (C6H11O4N)n, is naturally abundant in crustaceans, exoskeletons of insects, algaes, fungi and cytoderm of superior animals. In 1811, a French scientist Braconnot isolated a new substance from mushroom and denominated it chitin; Later in 1823, the same substance was also extracted from the shell of crustaceans by another French scientist and was given a new name as Fungine. Then in 1859, Lucheit obtained a new substance by treating chitin with sodium hydroxide at 120℃for 3 hours; Different to chitin, this substance was soluble in organic acids, and finally was denominated as chitosan by Annbosola in 1894.Researches about chitosan were carried out further deeply since chitosan was discovered. In 1977, the 1th International symposium about chitin and chitosan , at which the production,exploitation and utilization of chitin and chitosan was discussed for the first time, was hosted by Mit Sea Grand Program in Boston,US. Then, the 2th was held in Japan,1982; Recently in 1996 and 1999, symposium about chitin and chtosan was held subsepuently in Beijing and Wuhan, which deeply investigated and prospected the applications of chitosan in medicine and pharmacology,biology,food processing,textile industry,agriculture and environmental protection. With the development of science of macromolecule and biomedicine , the applications of chitosan get expanded and chitosan has been honoured the 6th element essential for life after glucose,protein,fat,vitamine and minerals for its extensive physiological functions.Pollution of environmental heavy metal of cadmium was persistent,hazardous and undegradable. The mutagenesis and carcinogenesis of cadmium pollution has provoked worldwide interest, which in turn make exploitation of antagonist and antidote of cadmium the focus of environmental health. Pro.Chen Xuemin , a Chinese specialist in the field of environmental hygiene, has established a series of methodology about heavy metals by a large number of studies such as the hydroxy free radical-scavenging effect of combined selenium and germanium, et al. But as antidotes of cadmium, the application of selenium and germanium was restricted severely because of the rather narrow safety dose range. In recent years, chitosan was found to be effective as adsorbent of heavy metals by Bhanoori and Gyliene, who have reported that chitosan could promote the metabolism and elimination of cadmium significantly. All of these studies have revealed the potential of chitosan as antidote of environmental heavy metals, especially cadmium.The scavenging effect of chitosan on hydroxy free radical produced in Fenton-reaction system has been testified by our former experiment. In this study, we employed cadmium as toxicant to induce oxidative damage of HepG2 cell, then investigated the role and mechanism of chitosan on cell viability,ROS level,oxidative damage of DNA and DNA repair.PartⅠ: The hydroxy free radical-scavenging effect of water-soluble chitosanPhenanthroline-Fe(Ⅱ) oxidation assay was adopted to detect the hydroxy free radical-scavenging effect of water-soluble chitosan in this study. In Fenton reaction system , ascorbic acid ( Vc ) was used as positive control , the hydroxy free radical-scavenging effect of water-soluble chitosan with different concentration was tested. It showed that: (1) the rate of hydroxy free radical-scavenging of water-soluble chitosan improved from 14.68% to 93.67% when the concentration of chitosan increased from 0.04mg/ml to 0.32 mg/ml; (2) The pseudo-positive scavenging effect on hydroxy free radical of water-soluble chitosan with the highest value of 9.03% was tested specially in the experiment. The rate of hydroxy free radical-scavenging of the chitosan was still up to 74.98%-84.64% when the rate of pseudo-positive scavenging was calibrated. The results demonstrated that water-soluble chitosan is an appropriate kind of hydroxy free radical-scavenging agent.PartⅡ: Mechanism of chitosan on the damage of HepG2 cell induced by cadmium chloride1. Effect of chitosan on the decrease of HepG2 cell viability induced by cadmium chloride .MTT Assay was employed to investigate the cytotoxic effect of cadmium chloride on human hepatocyte carcinoma cell (HepG2 cell) and antagonistic effect of chitosan on the decrease of HepG2 cell viability induced by cadmium chloride. The results showed that:(1) Cadmium caused decrease in cell viability in a time-dependent manner. Compared to normal control, cell viability decreased in 10μmol/L cadmium chloride group after exposured for 4, 8, 16 and 24h,respectively, but no significant difference was observed. In 20μmol/L and 30μmol/L cadmium chloride group, cell viability decreased significantly after exposured for 8,16 and 24h, respectively. In comparison with normal control, cell viability decreased with significant difference in 40μmol/L and 50μmol/L after exposured for 4, 8, 16 and 24h,respectively(P<0.05). (2) Compared to normal control, cell viability decreased in dose-dependent fashion with concentration of cadmium chloride. After exposured to cadmium chloride for 4h, cell viability decreased from 86.3% to 75.7% when concectration of cadmium chloride increased from 10μmol/L to 50μmol/L,but significant difference was only be observed in 40μmol/L and 50μmol/L group(P<0.05).In comparison with normal control,significant decrease of cell viability was observed in all but 10μmol/L cadmium treated group when exposure time was extended to 8, 16 and 24h, respectively(P<0.05).(3) Compared to normal control,cell viability decreased from 100% to 54.8% after exposured to 20μmol/L cadmium chloride for 24h.There is on significant difference between the cell viability of 0.50mg/ml chitsoan group and that of normal control. In experimental groups treated with chitosan and 20μmol/L cadmium chloride simultaneously, cell viability increased from 59.8% to 81.5% when chitosan concentratioin improved from 0.10mg/ml to 0.50 mg/ml. In comparison with 20μmol/L cadmium chloride group, significant increase of cell viability was observed in experimental group exposured to 0.50mg/ml chitosan and 20μmol/L cadmium chloride simultaneously.It has demonstrated that high dose of chitosan has no significant effect on viability of HepG2 cell, but has protect effect on HepG2 cell damage induced by cadmium chloride.2. The scavenging effect of chitosan on ROS in HepG2 cell induced by cadmium chloride.HepG2 cell was exposured to chitosan and cadmium chloride simultaneously for 4h.Two groups treated with cadmium chloride and chitosan respectively were used as control. The fluorescence of 2′,7′-dichlorofluorescin(DCF) was measured (by flowcytometry) as a mean of estimating the formation of reactive oxygen species (ROS).In comparison with normal control ,the DCF intensity of chitosan group only exhibited somewhat decrease(P>0.05) and a significant increase(P<0.01) was observed in cadmium chloride group(20μmol/L, 4h). The DCF intensity decreased in all three groups treated with cadmium chloride and chitosan(0.02, 0.10 and 0.50mg/ml, respectively) simultaneously are all higher than that of cadmium chloride-treated group, and when the concentration of chitosan improved from 0.01mg/ml to 0.50mg/ml, the DCF intensity decreased from 275.59 to 174.59 and significant decrease was observed at concentration of 0.50mg/ml. These results suggested that chitosan could antagonized the increase of ROS in HepG2 cell induced by cadmium chloride.3. The effect of chitosan on cadmium-induced DNA damage in HepG2 cell.In this part of study, we used single cell gel electrophoresis to measure DNA damaging effect caused by cadmium chloride in HepG2 cell and the effect of chitosan on DNA damage in HepG2 cell induced by cadmium chloride. The results showed that (1) cadmium chloride caused increase in Olive tail moment in a dose-dependent fashion. Statistical significant increases of Olive tail moment were observed in HepG2 cell treated with 20, 40 and 80μmol/L cadmium chloride for 4 hours. (2) The Olive tail moment of 0.50mg/ml chitosan group exhibited somewhat decrease in comparison with that of normal control, but no significant difference was observed. In the three groups treated with 20μmol/L cadmium chloride and chitosan(0.02, 0.10 and 0.50mg/ml,respectively) simultaneously, the Olive tail moment was 5.29, 5.11 and 1.61,respectively,which were all lower than that of 20μmol/L cadmium chloride-treated group, and significant decrease was observed in the group treated with cadmium and 0.50mg/ml chitosan simultaneously. These results demonstrated that chitosan was effective in protect HepG2 cell from DNA damage caused by cadmium chloride.4. The effect of chitosan on expression of hOGG-1 in oxidative damaged HepG2 cell induced by cadmium chloride.To make a further investigation on the effect of chitsoan on expression of hOGG-1 in cadmium chloride-induced HepG2 cell based on the studies on scavenging effect of cadmium-induced free radical with selenium and the hydroxy free radical- scavenging effect of water-soluble chitosan. HepG2 cell was treated with 40μmol/L cadmium chloride and chitosan simultaneously for 24h. 40μmol/L cadmium chloride and 0.50mg/ml were used as control,respectively. The expression of hOGG-1 was semi-quantitated by westernblotting. Results showed that: In comparison with normal control, the hOGG-1 content in 40μmol/L cadmium chloride group decreased significantly(P < 0.01). Compared to cadmium chloride-treated group, the hOGG-1 content were much higher in the groups treated with cadmium chloride(40μmol/L) and chitosan(0.02, 0.10 and 0.50mg/ml, respectively) simultaneously, and when the concentration of chtosan improved from 0.02mg/ml to 0.50 mg/ml, the hOGG-1 content increased and significant difference(p<0.01)was observed at the concentration of 0.50mg/ml .This study suggested that the decrease of hOGG-1 content in HepG2 cell induced by cadmium chloride could be antagonized effectively by chitosan .