Roles Of Reactive Oxygen Species In MAPKs Of Cells Exposed To Selenite

Selenium is an essential micronutrient with important biological functions. It plays a key role in the organism. The distribution of selenite is different in different area. Generally speaking, there are much selenite in tropical zone and subtroptical zone, but threre are little in temperate zone and prairie. An foreign investigation showed that cancer death rates were inversely correlated with the geographic distribution of Se in forage crops, and cancer of stomach, oesophagus and rectum were found to be particularly high in selenium-poor areas. A domestic investigation showed that the incidences of liver cancer were inversely correlated with Se in crops, and the distribution of Se was different in different area. Some epidemiological studies and experimental models have also indicated that high levels of Se compounds prevent cancer.There are some reports about the mechanism for the inhibited cancer of Se compounds, but it remains largely unclear about the mechanism of the cancer chemoprevention and inhibition. Now the mechanism may be the following reason: 1. Se compounds can inhibit cancer cells proliferation and promote them apoptosis. Various kinds of Se compounds can inhibit cells proliferation by decreasing the activity of CDC2+ and PKC. ROS can be induced by Se compounds reacted with GSH. Some apoptosis pathway is initiated, so cancer cells are induced apoptosis. 2. The mutagenicity of carcinogenic factor can be decreased by Se compounds. Se compounds decrease the activity of hydroxylase that can active carcinogen. Se compounds also can decrease the carcinogenicity of some carcinogenic metal. 3. By modulating immune system, Se compounds can enhance the anticancer ability. 4. The activity of some anti-oxidized enzyme can be modulated by Se compounds. Se compounds prevent lipid being oxidized and biomembrane being impaired, so the incidence of mutation can be decreased.The nutritional requirement of Se is small. The reports showed that toxic threshold level may be ten fold of nutritional requirement of Se, and human could be poisoned for ingesting thirty to fifty fold of nutritional requirement of Se. Now the mechanism of toxic effect is still not very clear. The two aspects are being considered as below: 1. ROS is thought being related with the toxicity of Se compounds. ROS can be induced by higher concentration of Se compounds, and it can react with biomacromolecule, including protein, DNA and lipid. Thus biomacromolecule are impaired. 2. Some metabolic enzyme can be inactive by Se compounds, so organism is damaged.In a word, no matter the anticancer ability or the toxicity of Se compounds is related with ROS. ROS is also closely related with some signal transduction pathway. Both exogenous and endogenous ROS can modulate the activity of MAPKs. MAPKs are regulated by distinct signal transduction pathways that control many aspects of mammalian cellular physiology, including cell growth, differentiation and apoptosis.In the present study, after Hepg2 cells were treated with selenite and male Wistar rat were administered by selenite, the DNA damage was measured by comet essays. The apoptosis rate and the levels of ROS were detected by flow cytometry. The levels of MAPK were determined by western-blot. Our study investigate how MAPKs are modulated by ROS.Part I The Investigation of Mechanisms about HepG2 Apoptosis Induced by SeleniteIn order to know how the cell viability is affected by selenite, after HepG2 was treated by selenite (0, 2.5, 5, 10, 20μmol/L) for 12, 24, 48 h, then the cell viability was detected. The results showed the cell viability decreases with the increase of time exposed to selentie and the concentration of selenite. Comparing with the control, the cell viability was decreased after HepG2 cells were treated with 10μmol/L selenite for 12 h or 5μmol/L selenite for 24 h (P<0.05). The cell viability was decreased after they are exposed to 10 or 20μmol/L selenite for 12, 24 and 48 h.For exploring how the level of ROS changed after HepG2 cells were treated by selenite, the level of ROS was measured by flow cytometry after the cells were treated by selenite (0, 2.5, 5, 10, 20μmol/L) for 0.5, 1, 2 h. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) readily diffuses through the cell membrane and is deacetylated by esterases to non-fluorescent 2′, 7′-dichoorofluorescin (DCFH). Further, DCFH could be rapidly oxidized to highly fluorescent DCF in the presence of ROS. The results showed that the increase in ROS was dose-dependent, and ROS increased significantly after cells exposed to 5, 10, or 20μmol/L for one hour. The level of ROS was increased in time-dependent manner, when cells were exposed to 10μmol/L of Se, and a significant increase in ROS was observed 30 min after the treatment with Se.The comet assays and flow cytometry were used to detect the DNA damage and apoptosis rate of cells after they are exposed to selenite. Chromosomal DNA strand breaks as measured by OTM value were increased in HepG2 cells exposed to different dose of Se (i.e., 5, 10, or 20μmol/L), compared with the controls of untreated HepG2 cells. No difference was observed in the group treated with 2.5μmol/L of Se and control group. After HepG2 cells were exposed to different doses (5, 10 and 20μmol/L) of Se for 24 hour, significant differences were observed between them and 0μmol/L Se. The percentage of early apoptosis and late apoptosis/necrosis increased from 1.11% to 16.60% and from 2.60% to 10.15%. After HepG2 were treated with 10μmol/L Se for 0 h, 12 h, 24 h and 48 h, the percentage of early apoptosis and late apoptosis/necrosis increased from 1.11% to 23.52% and from 2.60% to 14.80.In order to know the role of MAPKs in the apoptosis induced by selenite, the level of c-JUN N-terminal kinase, extracellular signal-regulated kinase and p38 were detected by western-blot after HepG2 were exposed to selenite (0, 2.5, 5, 10, 20μmol/L) for 4 h. Western blot analyses showed than the amounts of p38, phospho-p38, ERK1/2 and phospho-ERK1/2 were the same as in untreated cells, whereas the levels of phospho-JNK1/2 increased in a dose-dependent after HepG2 cells were exposed to Se for 4 h. Interestingly, the amounts of JNK2 increased with the doses of Se increasing, whereas levels of JNK1 were no difference between cells exposed to Se and untreated cells. The results showed changes in phospho-JNK1/2 in HepG2 cells with exposure to 10μmol/L Se for 0, 1, 2, 4 and 8 h. After an hour exposure to 10μmol/L Se, it caused increases in phospho-JNK1 about 1.58-fold and phospho-JNK2 about 1.80-fold above the control. After 4 h the increase in phospho-JNK1 was about 2.48-fold and phospho-JNK2 about 5.03-fold above the control. After 8 h, compared with cells treated with Se for 4 h, the levels of phospho-JNK1/2 decreased.Finally for further illuminating the role of ROS and JNK1/2 in the apoptosis induced by selenite, the viability, DNA damage, apoptosis rate and the level of JNK1/2 were detected after N-acetylcysteine (NAC), one of antioxidants, and sp600125, one special inhibitor of JNK, were treated with selenite. NAC increased cell viability and inhibited apoptosis and DNA damage in the cells exposed to Se. To confirm that the inhibition of apoptosis was associated with the antioxidant effects of NAC, the effects of NAC on the generation of ROS were examined. Our data showed that NAC indeed decreased DCF fluorescence, suggesting the increase of ROS was inhibited by NAC. Furthermore, western blot results show that NAC inhibited JNK1 phosphorylation induced by Se. Sp600125 decreased the rates of early apoptotic cell induction by 10μmol/L Se (P<0.05). Furthermore, sp600125 significantly diminished the activation of JNK1/2 when HepG2 exposed to 10μmol/L Se for 4 h (P<0.05).Part II A subchronic toxicity study of selenite in wistar ratIn order to explore the subchronic toxicity of selenite and the role of oxidative stress, 36 male wistar rats were randomly divided into 6 groups with 6 rats each group. The rats were administered saline or selenite at the dose of 0.125, 0.25, 0.5, 1, 2 mg/kg bw Se orally each day for 7 consecutive weeks. Body weights and feed consumption were measured. At the end of the test period, the rats were decapitated to obtain blood, l for clinical chemistry, selected organs were weighed and specified tissues from all animals were subsequent histopathological examination. The livers and kidneys were taken to prepare the single-cell suspensions for comet assay. Oliver tail moment (OTM) was used to evaluate DNA damage of cells from the rats.After 7 weeks, comparing with the control, the average body of the rats in 0.125mg/kg bw group increased, but the increase in not significant; the average body of the rats in 2 mg/kg bw group decreased (P<0.05). Comparing with the control, the relative liver in 1 and 2 mg/kg bw group increased (P<0.05), and the relative kidney and spleen in 2 mg/kg bw group increased (P<0.05), but no difference was seen in the relative heart and testis between different group.The results of pathological section showed hepatic cells presented hydropic degeneration, and a lot of vacuolation could be observed in 2mg/kg bw group. Hyperemia and hydropic change kidney could be observed in 2mg/kg bw group. With the increasing of doses of selenite, the level of alanine aminotransferase and aspartate amino transferase increased. Furthermore, the level of alanine aminotransferase and aspartate amino transferase is higher than the control (P<0.05), but no difference was observed in urea and creatinine of blood serum among different groups.We detected the level of glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) in blood serum and livers to explore the oxidative stress induced by selenite. Among different groups, no difference was observed in the level of SOD in blood serum and livers (P>0.05). The level of MDA in blood serum and livers in 2 mg/kg bw group was higher than in the control (P<0.05). The level of GSH in blood serum among 0.5, 1, 2 mg/kg bw groups is lower than in the control, however, only The level of GSH in liver in 1, 2 mg/kg bw groups is lower than the control.Comet assays was used to detect the DNA damage of liver and kidney cells induced by selenite. Comparing with the control, the results showed that the OTM of 0.5, 1, 2 mg/kg bw groups increased (P<0.05), and it indicated that selenite induced liver and kidney cells DNA damage.Conclusions:1) Se inhibited cells viability in a dose- and time-dependent manner. NAC could increase cell viability.2) HepG2 cells exposed to Se result in intracellular ROS increasing, and NAC can decrease the level of ROS induced by selenite.3) The dose of Se higher than 5μmol/L can induce apoptosis and DNA damage in HepG2, and NAC can inhibit the effects induced by selenite.4) The amounts of phospho-JNK1/2 increased with the doses of Se increasing. NAC inhibits JNK1 phosphorylation induced by Selenite. Sp600125 decreases the apoptosis induced by Selenite.5) 2 mg/kg selenite impairs the liver and kidney of the rats. The level of GSH in liver and blood serum decreases, and the level of MDA in liver and blood serum increases. Oxidative stress plays a key role in the damage induced by selenite.6) The results of comet assay show that DNA damage may be sensitive to the impairment induced by selenite. DNA damage may be the early evaluation indexes of the impairment induced by selenite.