硒诱导产生ROS在MAPKs信号转导通路中的调节作用
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摘要
硒是一种具有重要生物功能的必需微量元素,在生命活动中发挥着重要作用。硒在环境中的分布存在有明显的地域差异,一般热带以及亚热带含硒较高,而温带以及森林或草原地带含硒较低。国外有研究表明,一些肿瘤如胃癌、食道癌、直肠癌等的发病率和硒的地理分布呈负相关,低硒地区肿瘤的发病率和死亡率比较高。国内也有研究通过分析不同肝癌高发区的粮食硒的水平后,发现硒的分布有明显的地区差异,并且与肝癌的流行负相关。一些流行病学调查和动物实验也表明高浓度的硒具有化学防癌作用。
硒的抗肿瘤作用的机制有不少报道,但是总的来说,其防癌、抗癌作用机理目前还不是十分清楚。认为可能主要有以下几个方面:1.抑制肿瘤细胞增殖,促进肿瘤细胞凋亡。各种形式的硒均可以抑制CDC2+和PKC(蛋白激酶C)的活性,从而抑制细胞增殖;硒可以和还原性谷胱甘肽(GSH)反应产生活性氧(ROS),启动一些凋亡信号途径而促使肿瘤细胞凋亡。2.降低一些诱癌因子的诱变性。硒可以降低某些能激活致癌原的羟化酶如芳基羟化酶的活性;硒还可以和一些致癌性的金属离子相互作用,从而拮抗它们的活性。3.调节机体免疫系统,增强人体免疫系统的抗癌能力。4.调节一些生物体内抗氧化酶如谷胱甘肽过氧化酶的活性,防止脂质过氧化,保护生物膜不受损伤,防止突变。
另一方面,硒的生理需要量范围比较窄,有人认为硒的摄入量超过生理需要量10倍就可能达到中毒阈剂量水平,30～50倍可导致中毒。目前有关硒化合物的毒性作用机制也不是十分清楚,主要认为可能有以下两个方面:1.硒化合物的毒性和其催化产生的ROS有关。硒在较高浓度下产生ROS,它们可以和一些生物大分子包括蛋白质、DNA、脂质发生反应,从而使其受损。2.硒可使体内的一些代谢酶如琥珀酸脱氢酶等失活,从而使机体受损。
总而言之,无论是硒的抗肿瘤作用还是硒的毒性作用可能都和ROS有关。ROS和一些信号转导途径也密切相关,无论是内源性还是外源性的ROS都可以调节丝裂原活化蛋白激酶(MAPKs)的活性。不同的信号途径通过MAPKs调节细胞生理过程的很多方面,包括细胞增殖、分化和凋亡。
本研究用硒处理HepG2细胞以及给Wistar大鼠灌胃后,利用彗星实验、流式细胞术、Western-blot等方法观察细胞的DNA损伤和凋亡情况以及ROS和MAPK的表达水平,探讨硒染毒产生ROS在MAPKs信号转导通路中的调节作用。本研究共分两部分。
第一部分硒诱导HepG2细胞凋亡的作用机制的研究
首先为了了解不同浓度硒对细胞活性的影响,本研究应用MTT试验检测了不同剂量(0、2.5、5、10、20μmol/L)处理HepG2 (12、24、48 h)不同时间后的细胞活性。结果表明随着浓度以及作用时间的增加,亚硒酸钠对HepG2的抑制增殖作用越明显。10μmol/L亚硒酸钠作用12 h和5μmol/L亚硒酸钠作用24 h后细胞活性与对照组比较明显降低,差异有显著性(P<0.05)。10、20μmol/L的亚硒酸钠处理细胞后,在三个观察时间点(12、24、48 h)都可见细胞活性明显降低。
为了了解硒处理细胞后ROS的变化情况,本研究用流式细胞仪测定不同浓度硒(0、2.5、5、10、20μmol/L)处理细胞不同时间(0.5、1、2 h)后细胞内ROS的水平。ROS的测定是采用DCFH-DA,它本身不发荧光,进入细胞内后水解生成非荧光的DCFH,在ROS的存在下氧化成发荧光的DCF。结果显示5、10、20μmol/L亚硒酸钠作用于HepG2 lh,随着浓度增加,产生的ROS也增加。与对照组相比,当亚硒酸钠浓度≥5μmol/L时,流式峰明显右移,平均荧光强度增加(P<0.05)。10μmol/L亚硒酸钠处理HepG2细胞0.5 h后,即可以见到到荧光强度明显增加(P<0.05)。
为了阐明不同浓度硒处理细胞后其DNA损伤以及凋亡情况,我们通过彗星试验(单细胞琼脂糖凝胶电泳)、流式细胞术来检测细胞损伤以及凋亡情况。结果发现0、2.5、5、10、20μmol/L亚硒酸钠作用HepG2 24小时后,当亚硒酸钠浓度≥5μmol/L时,Olive尾矩明显增加,DNA损伤加重,与对照组相比有统计学意义(P<0.05)。流式细胞术检测结果表明0、2.5、5、10、20μmol/L亚硒酸钠处理HepG2 24 h后, 5、10、20μmol/L组早期凋亡细胞以及晚期凋亡细胞/坏死细胞均比对照组高((P<0.05)。随亚硒酸钠剂量从0μmol/L增加到20μmol/L,早期凋亡率和晚期凋亡/坏死细胞率分别从1.11%、2.60%增加到16.6%、10.15%。10μmol/L亚硒酸钠处理HepG2 0、12、24、48 h后,早期凋亡和晚期凋亡/坏死细胞分别从1.11%、2.60%增加到23.52%、14.8%。
为了了解MAPKs在亚硒酸钠导致细胞凋亡中的可能作用,我们用不同浓度(0、2.5、5、10、20μmol/L)亚硒酸钠处理HepG2细胞4 h后,使用Western-blot技术检测c-Jun氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)以及p38的表达水平。结果表明各处理组p38、磷酸化p38、ERK1/2和磷酸化ERK1/2水平和对照组无明显差异,而磷酸化JNK1/2的水平随着亚硒酸钠的剂量的增加而增加。10μmol/L亚硒酸钠处理HepG2细胞0、1、2、4、8 h后,用Western-blot技术检测磷酸化的JNK1/2水平。结果1 h后磷酸化的JNK1和JNK2分别为对照的1.58和1.80倍;4 h后磷酸化的JNK1和JNK2分别为对照的2.48和5.03倍;8 h后,和亚硒酸钠处理4 h相比,磷酸化的JNK1/2水平下降。
最后为了进一步阐明ROS以及JNK1/2在细胞凋亡中的可能作用以及ROS对JNK1/2的影响,我们使用ROS清除剂N-乙酰半胱氨酸(NAC)后来看亚硒酸钠处理细胞对细胞活性、DNA损伤程度、凋亡情况以及JNK1/2表达水平的影响,以及使用JNK1/2抑制剂sp600125后来看凋亡情况以及JNK1/2表达水平的变化。结果表明NAC增加了细胞活性并降低了细胞凋亡率以及DNA损伤程度,并且NAC处理的亚硒酸钠组ROS水平下降,Western-blot结果表明NAC抑制了JNK1的磷酸化。和10μmol/L亚硒酸钠单独作用组比较,Sp600125预处理的10μmol/L亚硒酸钠组磷酸化的JNI1/2水平降低(P<0.05),并且与对照组比较,细胞早期凋亡率也下降(P<0.05)。
第二部分硒亚急性染毒对wistar大鼠的影响
为了阐明硒的毒作用表现及氧化应激在其中所起的作用,我们将36只雄性wistar大鼠随机分成6组,各组剂量分别为0、0.125、0.25、0.5、1、2mg/kg体重,经口灌胃7周,每天同一时间灌胃一次.每周记录一次大鼠体重以及饲料消耗量,染毒结束后断头取血检测临床生化指标,取肝、肾等组织称重、做病理切变并用彗星实验分析其DNA损伤情况.
结果表明,染毒七周后,和对照组相比,0.125mg/kg组大鼠的平均体重增加,但是经检验无统计学意义; 2mg/kg亚硒酸钠组,7周后体重降低(P<0.05)。脏器系数分析结果表明,与对照组相比,1mg/kg和2mg/k亚硒酸钠组,肝脏的相对重量增加(P<0.05);2mg/kg亚硒酸钠组,肾脏和脾脏的相对重量增加(P<0.01)。对心脏和睾丸则未见影响,各组之间未见差异(P>0.05)。
为了检测不同亚硒酸钠浓度对肝肾功能的影响,对肝肾组织做了病理切片并检测了一些临床生化指标。病理切片分析结果显示2mg/kg组肝细胞水肿、变性,组织呈大片空泡样细胞,其它各组未见明显变化;肾组织可见充血、水肿,其它与对照组比较无明显差异。生化分析结果表明随亚硒酸钠剂量增加,丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)均增加,与对照组相比,1mg/kg和2mg/kg亚硒酸钠组,谷丙转氨酶(ALT)和谷草转氨酶(AST)均增加明显(P<0.05)。而各组之间血清尿素和肌酐未见明显差异(P>0.05)。
为了进一步了解亚硒酸钠染毒产生ROS所造成的氧化应激,我们检测了血清和肝组织的还原性谷胱甘肽(GSH)、超氧化物歧化酶(SOD)酶活性以及脂质过氧化产物丙二醛(MDA)的含量.结果显示血清和肝组织各组间SOD活性无明显差异(P>0.05);血清和肝组织的丙二醛(MDA)含量除了2mg/kg亚硒酸钠组与对照组相比明显升高(P<0.05),其余各组和对照组之间无明显差异(P>0.05);血清GSH在0.5、1、2mg/kg亚硒酸钠组和对照组相比明显降低(P<0.05),且有随剂量增加而降低的趋势,肝组织GSH在1、2mg/kg亚硒酸钠组和对照组相比明显降低(P<0.05)。
我们通过彗星实验进一步研究亚硒酸钠对大鼠肝、肾的DNA损伤情况。实验结果显示和对照组相比较,0.5、1、2mg/kg亚硒酸钠组肝、肾细胞OTM值增加,表明DNA损伤增加(P<0.05)。
综上所述,本研究结果表明:
(1)亚硒酸钠可抑制HepG2细胞活性,且具有剂量和时间依赖性,NAC可以拮抗亚硒酸钠对细胞活性的抑制作用。
(2)亚硒酸钠可使HepG2细胞内的ROS增加,而NAC可以使亚硒酸钠诱导的ROS下降.
(3)亚硒酸钠可导致HepG2细胞DNA受损,细胞凋亡增加,NAC可以使亚硒酸钠导致的细胞DNA受损程度降低,并减少细胞凋亡。
(4)亚硒酸钠可激活JNK1/2,NAC和sp600125可使JNK1/2磷酸化激活受到抑制,并且sp600125降低了亚硒酸钠导致的细胞凋亡。
(5) 2 mg亚硒酸钠/kg体重亚急性染毒主要导致大鼠肝、肾受损,尤其是肝脏,血清和肝组织的GSH降低,MDA增加,机体的氧化平衡受到破坏可能在亚硒酸钠的毒性中发挥了重要作用。
(6)彗星实验表明DNA损伤可能是亚硒酸钠导致损害比较敏感的指标,可以考虑作为一个亚硒酸钠导致机体损伤的早期评价指标。
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 Selenite
In 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 rat
In 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.
硒的抗肿瘤作用的机制有不少报道,但是总的来说,其防癌、抗癌作用机理目前还不是十分清楚。认为可能主要有以下几个方面:1.抑制肿瘤细胞增殖,促进肿瘤细胞凋亡。各种形式的硒均可以抑制CDC2+和PKC(蛋白激酶C)的活性,从而抑制细胞增殖;硒可以和还原性谷胱甘肽(GSH)反应产生活性氧(ROS),启动一些凋亡信号途径而促使肿瘤细胞凋亡。2.降低一些诱癌因子的诱变性。硒可以降低某些能激活致癌原的羟化酶如芳基羟化酶的活性;硒还可以和一些致癌性的金属离子相互作用,从而拮抗它们的活性。3.调节机体免疫系统,增强人体免疫系统的抗癌能力。4.调节一些生物体内抗氧化酶如谷胱甘肽过氧化酶的活性,防止脂质过氧化,保护生物膜不受损伤,防止突变。
另一方面,硒的生理需要量范围比较窄,有人认为硒的摄入量超过生理需要量10倍就可能达到中毒阈剂量水平,30～50倍可导致中毒。目前有关硒化合物的毒性作用机制也不是十分清楚,主要认为可能有以下两个方面:1.硒化合物的毒性和其催化产生的ROS有关。硒在较高浓度下产生ROS,它们可以和一些生物大分子包括蛋白质、DNA、脂质发生反应,从而使其受损。2.硒可使体内的一些代谢酶如琥珀酸脱氢酶等失活,从而使机体受损。
总而言之,无论是硒的抗肿瘤作用还是硒的毒性作用可能都和ROS有关。ROS和一些信号转导途径也密切相关,无论是内源性还是外源性的ROS都可以调节丝裂原活化蛋白激酶(MAPKs)的活性。不同的信号途径通过MAPKs调节细胞生理过程的很多方面,包括细胞增殖、分化和凋亡。
本研究用硒处理HepG2细胞以及给Wistar大鼠灌胃后,利用彗星实验、流式细胞术、Western-blot等方法观察细胞的DNA损伤和凋亡情况以及ROS和MAPK的表达水平,探讨硒染毒产生ROS在MAPKs信号转导通路中的调节作用。本研究共分两部分。
第一部分硒诱导HepG2细胞凋亡的作用机制的研究
首先为了了解不同浓度硒对细胞活性的影响,本研究应用MTT试验检测了不同剂量(0、2.5、5、10、20μmol/L)处理HepG2 (12、24、48 h)不同时间后的细胞活性。结果表明随着浓度以及作用时间的增加,亚硒酸钠对HepG2的抑制增殖作用越明显。10μmol/L亚硒酸钠作用12 h和5μmol/L亚硒酸钠作用24 h后细胞活性与对照组比较明显降低,差异有显著性(P<0.05)。10、20μmol/L的亚硒酸钠处理细胞后,在三个观察时间点(12、24、48 h)都可见细胞活性明显降低。
为了了解硒处理细胞后ROS的变化情况,本研究用流式细胞仪测定不同浓度硒(0、2.5、5、10、20μmol/L)处理细胞不同时间(0.5、1、2 h)后细胞内ROS的水平。ROS的测定是采用DCFH-DA,它本身不发荧光,进入细胞内后水解生成非荧光的DCFH,在ROS的存在下氧化成发荧光的DCF。结果显示5、10、20μmol/L亚硒酸钠作用于HepG2 lh,随着浓度增加,产生的ROS也增加。与对照组相比,当亚硒酸钠浓度≥5μmol/L时,流式峰明显右移,平均荧光强度增加(P<0.05)。10μmol/L亚硒酸钠处理HepG2细胞0.5 h后,即可以见到到荧光强度明显增加(P<0.05)。
为了阐明不同浓度硒处理细胞后其DNA损伤以及凋亡情况,我们通过彗星试验(单细胞琼脂糖凝胶电泳)、流式细胞术来检测细胞损伤以及凋亡情况。结果发现0、2.5、5、10、20μmol/L亚硒酸钠作用HepG2 24小时后,当亚硒酸钠浓度≥5μmol/L时,Olive尾矩明显增加,DNA损伤加重,与对照组相比有统计学意义(P<0.05)。流式细胞术检测结果表明0、2.5、5、10、20μmol/L亚硒酸钠处理HepG2 24 h后, 5、10、20μmol/L组早期凋亡细胞以及晚期凋亡细胞/坏死细胞均比对照组高((P<0.05)。随亚硒酸钠剂量从0μmol/L增加到20μmol/L,早期凋亡率和晚期凋亡/坏死细胞率分别从1.11%、2.60%增加到16.6%、10.15%。10μmol/L亚硒酸钠处理HepG2 0、12、24、48 h后,早期凋亡和晚期凋亡/坏死细胞分别从1.11%、2.60%增加到23.52%、14.8%。
为了了解MAPKs在亚硒酸钠导致细胞凋亡中的可能作用,我们用不同浓度(0、2.5、5、10、20μmol/L)亚硒酸钠处理HepG2细胞4 h后,使用Western-blot技术检测c-Jun氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)以及p38的表达水平。结果表明各处理组p38、磷酸化p38、ERK1/2和磷酸化ERK1/2水平和对照组无明显差异,而磷酸化JNK1/2的水平随着亚硒酸钠的剂量的增加而增加。10μmol/L亚硒酸钠处理HepG2细胞0、1、2、4、8 h后,用Western-blot技术检测磷酸化的JNK1/2水平。结果1 h后磷酸化的JNK1和JNK2分别为对照的1.58和1.80倍;4 h后磷酸化的JNK1和JNK2分别为对照的2.48和5.03倍;8 h后,和亚硒酸钠处理4 h相比,磷酸化的JNK1/2水平下降。
最后为了进一步阐明ROS以及JNK1/2在细胞凋亡中的可能作用以及ROS对JNK1/2的影响,我们使用ROS清除剂N-乙酰半胱氨酸(NAC)后来看亚硒酸钠处理细胞对细胞活性、DNA损伤程度、凋亡情况以及JNK1/2表达水平的影响,以及使用JNK1/2抑制剂sp600125后来看凋亡情况以及JNK1/2表达水平的变化。结果表明NAC增加了细胞活性并降低了细胞凋亡率以及DNA损伤程度,并且NAC处理的亚硒酸钠组ROS水平下降,Western-blot结果表明NAC抑制了JNK1的磷酸化。和10μmol/L亚硒酸钠单独作用组比较,Sp600125预处理的10μmol/L亚硒酸钠组磷酸化的JNI1/2水平降低(P<0.05),并且与对照组比较,细胞早期凋亡率也下降(P<0.05)。
第二部分硒亚急性染毒对wistar大鼠的影响
为了阐明硒的毒作用表现及氧化应激在其中所起的作用,我们将36只雄性wistar大鼠随机分成6组,各组剂量分别为0、0.125、0.25、0.5、1、2mg/kg体重,经口灌胃7周,每天同一时间灌胃一次.每周记录一次大鼠体重以及饲料消耗量,染毒结束后断头取血检测临床生化指标,取肝、肾等组织称重、做病理切变并用彗星实验分析其DNA损伤情况.
结果表明,染毒七周后,和对照组相比,0.125mg/kg组大鼠的平均体重增加,但是经检验无统计学意义; 2mg/kg亚硒酸钠组,7周后体重降低(P<0.05)。脏器系数分析结果表明,与对照组相比,1mg/kg和2mg/k亚硒酸钠组,肝脏的相对重量增加(P<0.05);2mg/kg亚硒酸钠组,肾脏和脾脏的相对重量增加(P<0.01)。对心脏和睾丸则未见影响,各组之间未见差异(P>0.05)。
为了检测不同亚硒酸钠浓度对肝肾功能的影响,对肝肾组织做了病理切片并检测了一些临床生化指标。病理切片分析结果显示2mg/kg组肝细胞水肿、变性,组织呈大片空泡样细胞,其它各组未见明显变化;肾组织可见充血、水肿,其它与对照组比较无明显差异。生化分析结果表明随亚硒酸钠剂量增加,丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)均增加,与对照组相比,1mg/kg和2mg/kg亚硒酸钠组,谷丙转氨酶(ALT)和谷草转氨酶(AST)均增加明显(P<0.05)。而各组之间血清尿素和肌酐未见明显差异(P>0.05)。
为了进一步了解亚硒酸钠染毒产生ROS所造成的氧化应激,我们检测了血清和肝组织的还原性谷胱甘肽(GSH)、超氧化物歧化酶(SOD)酶活性以及脂质过氧化产物丙二醛(MDA)的含量.结果显示血清和肝组织各组间SOD活性无明显差异(P>0.05);血清和肝组织的丙二醛(MDA)含量除了2mg/kg亚硒酸钠组与对照组相比明显升高(P<0.05),其余各组和对照组之间无明显差异(P>0.05);血清GSH在0.5、1、2mg/kg亚硒酸钠组和对照组相比明显降低(P<0.05),且有随剂量增加而降低的趋势,肝组织GSH在1、2mg/kg亚硒酸钠组和对照组相比明显降低(P<0.05)。
我们通过彗星实验进一步研究亚硒酸钠对大鼠肝、肾的DNA损伤情况。实验结果显示和对照组相比较,0.5、1、2mg/kg亚硒酸钠组肝、肾细胞OTM值增加,表明DNA损伤增加(P<0.05)。
综上所述,本研究结果表明:
(1)亚硒酸钠可抑制HepG2细胞活性,且具有剂量和时间依赖性,NAC可以拮抗亚硒酸钠对细胞活性的抑制作用。
(2)亚硒酸钠可使HepG2细胞内的ROS增加,而NAC可以使亚硒酸钠诱导的ROS下降.
(3)亚硒酸钠可导致HepG2细胞DNA受损,细胞凋亡增加,NAC可以使亚硒酸钠导致的细胞DNA受损程度降低,并减少细胞凋亡。
(4)亚硒酸钠可激活JNK1/2,NAC和sp600125可使JNK1/2磷酸化激活受到抑制,并且sp600125降低了亚硒酸钠导致的细胞凋亡。
(5) 2 mg亚硒酸钠/kg体重亚急性染毒主要导致大鼠肝、肾受损,尤其是肝脏,血清和肝组织的GSH降低,MDA增加,机体的氧化平衡受到破坏可能在亚硒酸钠的毒性中发挥了重要作用。
(6)彗星实验表明DNA损伤可能是亚硒酸钠导致损害比较敏感的指标,可以考虑作为一个亚硒酸钠导致机体损伤的早期评价指标。
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 Selenite
In 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 rat
In 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.
引文
1 Shamberger R J, Frost DV, Possible protective effect of selenium against human cancer. Can Med Assoc J, 1969; 100:682.
2 Ip C, Lisk D J. Efficacy of cancer prevention by high-selenium garlic is primarily dependent on the action of selenium. Carcinogenesis, 1995; 16:2649–2652.
3 Clark LC, Combs GF Jr, Turnbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA, 1996; 1276:1957-1963.
4 Duffield-Lillico AJ, Reid ME, Turnbull BW, et al. Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer Epidemiol Biomarkers Prev, 2002; 11:630-6.
5 Corcoran NM, Najdovska M, Costello AJ. Inorganic selenium retards progression of experimental hormone refractory prostate cancer. J Urol, 2004; 171:907-910.
6 Zhuo HJ, Smith, AH, Steinmaus C. Selenium and lung cancer: a quantitative analysis of heterogeneity in the current epidemiological literature. Cancer Epidemiol Biomarkers Prev, 2004; 13:771-778.
7 Li L, Xie Y, El-Sayed WM, et al. Chemopreventive activity of selenocysteine prodrugs against tobacco-derived nitrosamine (NNK) induced lung tumors in the A/J mouse. J Biochem Mol Toxicol, 2005; 19:396-405.
8 El-bayoumy, K. The protective role of selenium on genetic damage and on cancer. Mutat Res, 2001, 475:123-139
9 Carson, D.A. Ribeiro, J.M. Apotosis and disease, Lancet, 1993; 341:1251-1254.
10 Stewart MS, Davis RL, Walsh LP, et al. Induction of differentiation and apoptosis by sodium selenite in human colonic carcinoma cells (HT29). Cancer Lett, 1997; 117:35-40.
11 Shen HM, Yang CF, Ong CN. Sodium selenite-induced oxidative stress and apoptosis in human hepatoma HepG2 cells. Int J Cancer, 1999; 81:820-828.
12 Jung U, Zheng X, Yoon SO, et al. Se-Methylselenocysteine induces apoptosismediated by reactive oxygen species in HL-60 cells. Free Radic Biol Med, 2001; 31:479-489.
13 Wang H Yang X Zhang Z, et al. Both calcium and ROS as common signals mediate selenite-induced apoptosis in SW480 colonic carcinoma cells. J Inorg Biochem, 2003; 97:221-230.
14 Cao TM, Hua FY, Xu CM, et al. Distinct effects of different concentrations of sodium selenite on apoptosis, cell cycle, and gene expression profile in acute promyeloytic leukemia-derived NB4 cells. Ann Hematol, 2006; 85:434-442.
15 Zhou N, Xiao H, Li TK, et al. DNA Damage-mediated Apoptosis Induced by Selenium Compounds. J Biol Chem, 2003; 278:29532-29537.
16 Zhong W, Oberley TD. Redox-mediated effects of selenium on apoptosis and cell cycle in the LNCaP human prostate cancer cell line. Cancer Res, 2001; 61:7071-7078.
17 Zuo L, Li J, Yang Y, et al. Sodium selenite induces apoptosis in acute promyelocytic leukemia-derived NB4 cells by a caspase-3-dependent mechanism and a redox pathway different from that of arsenic trioxide. Ann Hematol, 2004; 83:751-758.
18 Hu D, Liu Q, Cui H, et al. Effects of amino acids from selenium-rich silkworm pupas on human hepatoma cells. Life Sci, 2005; 77:2098-2110.
19 Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol, 1998; 10:248-253.
20 Dalton TP, Shertzer HG, Puge A. Regulation of gene expression by reactive oxygen. Pharmacol Toxicol, 1999; 39:67-101.
21 Hancock JT, Desikan R, Neill SJ. Role of reactive oxygen species in cell signalling pathways. Biochem Soc Trans, 2001; 29:345-350.
22 Kyriakis JM, Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays, 1996; 18:567-577.
23 Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med, 2000; 29:323-333.
24 Droge w. Free radicals in the physiological control of cell function. Physiol Rev, 2002; 82:47-95.
25 Torres M. Mitogen-activated protein kinase pathways in redox signaling. Front Biosci, 2003; 8:369-391.
26郭海恩,董力,余淑懿,等。硼硒联合对大鼠的急性和亚急性毒性研究。卫生研究,2002; 31(4):297-298.
27 Stacey NH, Klaassen CD. Comparison of the effects of metals on cellular injury and lipid peroxidation in isolated hepatocytes. J Toxicol environ Health, 1981; 7: 139–147.
28 Dougherty JJ, Hoekstra WG. Stimulation of lipid peroxidation in vivo by injected selenite and lack of stimulation by selenate. Proc Soc exp Biol Med, 1982; 169: 209–215.
29 Yan L, Yee JA, Boylan LM, et al. Effect of selenium compounds and thiols on human mammary tumor cells. Biol Trace Elem Res, 1991; 30:145–162.
30 Spallholz JE. On the nature of selenium toxicity and carcinostatic activity. Free Radicals Biol Med, 1994; 17:45–64.
31 Hu ML, Spallholz JE. In vitro hemolysis of rat erythrocytes by selenium compounds. Biochem Pharmacol, 1983; 32(6):957-61.
32 Hoffman JL. Selenite toxicity, depletion of liver S-adenosylmethionine, and inactivation of methionine adenosyltrasferase. Arch Biochem Biophys, 1977; 179(1):136-40.
33 Kajander EQ, Harvima RJ, Eloranta TO, et al. Metabolism, cellular actions, and cytotoxicity of selenomethionine in cultured cells. Biol Trace Elem Res, 1991; 28(1):57-68.
34 Ronai Z, Tillotson JK, Traganos F, et al. Effects of organic and inorganic selenium compounds on rat mammary tumor cells. Int J Cancer. 1995; 63(3):428-34.
35 Nano JL, Czerucka D, Menguy F, et al. Effect of selenium on the growth of three human colon cancer cell lines. Biol trance Elem Res, 1989; 20:31-43.
36 Kuchan MJ, Milner JA. Influence of intracellular glutathione on selenite-mediated growth inhibition of canine mammary tumor cells. Cancer Res, 1992; 52:1091-1095.
37 Greeder GA, Milner JA. Factors influencing the inhibitory effect of selenium on mice inoculated with Ehrlich ascites tumor cells. Science, 1980; 209:825-827.
38 Milner JA. Effect of selenium on virally induced and transplantable tumor models. Fed Proc, 1985; 44-2568-2572.
39 Medina D, Lane HW, Tracey CM. Selenium and mouse mammary tumorigenesis: aninvestigation of possible mechanisms. Cancer Res, 1983, 43:2460-2464.
40 Ip C, Lisk DJ. Efficacy of cancer prevention by high-selenium garlic is primarily dependent on the action of selenium. Carcinogenesis, 1995; 16:2649–2652.
41 Thompson HJ, Wilson A, Lu J, et al. Comparison of the effects of an organic and an inorganic form of selenium on a mammary carcinoma cell line. Carcinogenesis, 1994; 15:183-186
42 Ronai Z, Tillotson JK, Traganos F, et al. Effects of organic and inorganic selenium compounds on rat mammary tumor cells. Int J Cancer, 1995; 63:428-434.
43 Sinha R, Medina D. Inhibition of cdk2 kinase activity by methylselenocysteine in synchronized mouse mammary epithelial tumor cells. Carcinogenesis, 1997; 18:1541–1547.
44 Gopee NV, Johnson VJ, Sharma RP. Sodium selenite-induced apoptosis in murine B-lymphoma cells is associated with inhibition of protein kinase C-delta, nuclear factor kappaB, and inhibitor of apoptosis protein Toxicol Sci, 2004; 78:204-214
45张保国,杨劲松,姚乐申,沈苏南。硒酸钠对人前列腺癌细胞PC-3细胞活力和增殖的影响。中国癌症杂志,2005; 15(2):153-155.
46 Zhu, Z; Jiang W, Ganther HE, et al. Activity of Se-allylselenocysteine in the presence of methionine gamma-lyase on cell growth, DNA integrity, apoptosis, and cell-cycle regulatory molecules. Mol Carcinog, 2000, 29 (4):191–197
47 Seko Y, Saito Y, Kitahara J, et al. Active oxygen generation by the reaction of selenite with reduced glutathione in vitro. In A. Wendel (ed.), Selenium in biology and medicine. 1989; 70–73.
48 Sundaresan M, Yu ZX, Ferrans VJ, et al. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science, 1995; 270: 296-299.
49 Combs GF Jr, Gray WP. Chemopreventive agents: selenium. Pharmacol Ther, 1998; 79:179–192
50 Ganther HE. Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis, 1999; 20: 1657–1666.
51 Harrison PR, Lanfea J, Wu L, et al. Chemopreventive and growth inhibitory effects ofselenium. Biomed Environ Sci, 1997; 10, 235–245.
52 Stewart M S, Spallholz JE, Neldne, K H, et al. Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis. Free Radic Bio. Med, 1999; 26, 42–48
53朱志良,庄志雄,黄钰,等.单细胞凝胶电泳图像分析系统的研制及应用.中华劳动卫生职业病杂志, 2001; 19(4):298-300.
54 Olive PL, Banath JP, Durand RE. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the“comet”assay . Radiat Res, 1990; 122(1):86-94.
55 Tang R, Liu H, Wang T, et al. Mechanisms of selenium inhibition of cell apoptosis induced by oxysterols in rat vascular smooth muscle cells. Arch Biochem Biophys, 2005; 441(1):16-24.
56 Drake EN. Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant. Med Hypotheses, 2006; 67 (2): 318-322.
57 Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol. Today, 1994; 15:7–10.
58 Slater, AF, Nobel CS, Orrenius S. The role of intracellular oxidants in apoptosis. Biochim Biophy. Acta, 1995; 1271:59–62.
59 Kamata H, Hirata H. Redox regulation of cellular signalling. Cell Signal, 1999; 11:1–14.
60 Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol, 1999; 57:231–245.
61 Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J. Immunol Methods, 1995; 184:39-51.
62 Xing C, Peng Y, Chang R, et al. Effects of insulin-like growth factor-1 on okadaic acid-induced apoptosis in SH-SY5Y cells. Cell Biol. Int, 2005; 29:803-808.
63 Kramer GF, Ames BN. Mechanisms of mutagenicity and toxicity of sodium selenium selenite (Na2SeO3) in Salmonella typhimurium. Mutat Res, 1988; 201 (1):169-180
64 Jiang C, Hu H, Malewicz B, et al. Selenite-induced p53 Ser-15 phosphorylation andcaspase-mediated apoptosis in LNCaP human prostate cancer cells. Mol Cancer Ther. Mol Cancer Ther, 2004; 3(7):877-884.
65 Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell, 1995; 80:179-185.
66 Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med, 1996; 74:589-607.
67 Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal Kinase (JNK)-from inflammation to development. Curr Opin Cell Biol, 1998; 10:205-219.
68 Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell, 1995; 80:199-211.
69 Xia Z, Dickens M, Raingeaud, J, et al. Opposing effects of ERK and JNK-p38 MAP kinases on aopotosis. Science, 1995; 270:1326-1331.
70 Johnson NL, Gardner AM, Diner KM, et al. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. J Biol Chem, 1996; 271:3229-3237.
71 Zanke BW, Boudreau K, Rubie E, et al. The stress-activated protein kinase pathways mediate cell death following injury induced by cisplatinum, UV irradiation or heat. Curr Biol, 1996; 6:606-613.
72 Goillot E, Raingeaud J, Ranger A, et al. Mitogen-activated protein kinase -mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA, 1997; 94:3302-3307.
73 Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science, 1997; 275:90-94.
74 Rincon M, Whitmarsh A, Yang DD, et al. The JNK pathway regulates the in vivo deletion of immature CD4(+)CD8(+) thymocytes. J Exp Med, 1998; 188:1817 -1830.
75 Laszlo A. Evidence for two states of thermotolerance in mammalian cells. Int J Hyperthermia, 1988; 4(5):513-526.
76 Currie RW, Tanguay RM, Kingma JG, et al. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation, 1993; 87(3):963-971.
77 Mittler R, Berkowitz G. Hydrogen peroxide, a messenger with too many roles? Redox Rep. 2001; 6:69– 72.
78 Sata N, Klonowski-Stumpe H, Han B, et al. Menadione induces both necrosis and apoptosis in rat pancreatic acinar AR4-2J cells. Free Radic. Biol. Med, 1997; 23:844–850.
79 Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J. Cell Physiol, 2002; 192:1–15.
80 Ding M, Li J, Leonard SS, Shi X, et al. Differential role of hydrogen peroxide in UVinduced signal transduction. Mol. Cell Biochem, 2002; 234– 235:81– 90.
81 Chan WH, Wu CC, Yu JS. Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells. J. Cell. Biochem, 2003; 90:327– 338.
82 Lee SA, Dritschilo A, Jung M. Role of ATM in oxidative stressmediated c-Jun phosphorylation in response to ionizing radiation and CdCl2. J. Biol. Chem, 2001; 276:11783–11790.
83 Yu C, Rahmani M, Dent, P, Grant S. The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib. Exp. Cell Res, 2004; 295:555–566.
84 Mansat-de M V, Bezombes C, Quillet-Mary A, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol, 1999; 56:867– 874.
85 Mansat-de MV, Bezombes C, Quillet-Mary A, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol, 1999; 56:867– 874.
86 Shakibaei M, Schulze-Tanzil G, Takada Y, et al. Redox regulation of apoptosis by members of the TNF superfamily. Antioxid.Redox Signaling, 2005; 7:482– 496.
87 Goossens V, DeVos K, Vercammen D, et al. Redox regulation of TNF signaling. Biofactors, 1999; 10:145– 156.
88 Garg AK, Aggarwal BB. Reactive oxygen intermediates in TNF signaling. Mol. Immunol, 2002; 39:509– 517.
89 Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Sicence; 1997; 275:90-94.
90 Yoshizumi M, Abe J, Haendeler J, et al. Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem, 20002;
75:11706-11712.
91 Li Y, Arita Y, Koo HC, Davis JM, et al. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003; 29:779-783.
92 O’Tool D, Raisbeck M, Case JC, et al. Selenium-induced bblind staggersQ and related myths. A commentary on the extent of historical livestock losses attributed to selenosis on Wester US rangelands. Vet. Pathol, 1996; 33:104–116.
93 Oldfield JE. The two faces of selenium. J Nutr, 1987; 117:2002–2008.
94 Copper WC, Glover JR. 1974. The toxicology of selenium and its compounds. Van Nostrand Reinhold, New York, pp. 654–674.
95 Kramer G.F, Ames BN. Mechanisms of mutagenicity and toxicity of sodium selenite (Na2SeO3) in Salmonella typhimurium. Mutat Res, 1988; 201:169–180.
96 Seko Y, Imura N. Active oxygen generation as a possible mechanism of selenium toxicity, Biomed Environ Sci, 1997; 10:333–339.
97 Spallholz E. Free radical generation by selenium compounds and their prooxidant toxicity. Biomed Environ Sci, 1997; 10:260–270.
98 Spallholz JE, Palace VP, Reid TW. Methioninase and selenomethionine but not Se-methylselenocysteine generate methylselenol and superoxide in an in vitro chemiluminescent assay: implications for the nutritional carcinostatic activity of selenoamino acids. Biochem. Pharmacol, 2004; 67:547–554.
99 Terada A, Yoshida M, Seko Y, et al. Active oxygen species generation and cellular damage by additives of parenteral preparations: selenium and sulfhydryl compounds. Nutrition, 1999; 15:651–655.
100 Lu J, Kaeck M, Jiang C, et al. Thompson, Selenite induction of DNA strand breaks and apoptosis in mouse leukemic L1210 cells. Biochem Pharmacol, 1994; 47:1531–1535.
101 Ip C. Lessons from basic research in selenium and cancer prevention. Journal of Nutrition, 1998; 128:1845–1854.
102 Thompson HJ, Meeker LD, Becci PJ. Effect of combined selenium and retinyl acetate treatment on mammary carcinogenesis. Cancer Res, 1981; 41:1413–1416.
103 Temple N.J Basu TK. Selenium and cabbage and colon carcinogenesis in mice. J Natl Cancer Inst, 1987; 79:1131–1134.
104 Yan L, Yee JA, Li D, et al. Dietary supplementation of selenomethionine reduces metastasis of melanoma cells in mice. Anticancer Research, 1999; 19:1337–1342.
105 Tanaka T, Kohno H, Murakami M, et al. Suppressing effects of dietary supplementation of the organoselenium 1,4-phenylenebis(methylene)selenocyan -ate and the Citrus antioxidant auraptene on lung metastasis of melanoma cells in mice. Cancer Research, 2000; 60:3713–3716.
106 Thorlacius-Ussing O. Selenium-induced growth retardation. Histochemical and endocrinological studies on the anterior pituitaries of selenium treated rats. Danish Medical Bulletin, 1990; 37:347–358.
107 Orskov H, Flyvbjerg A. Selenium and human health. Lancet, 2000; 356:942–943.
108 Nakamuro K, Sayato Y, Tonomura M, et al. Studies on selenium related compounds. III. Acute and subacute toxicity of sodium selenate. Eisei Kagaku, 1974; 18:29–35.
109 Jia X, Li N, Chen J. A subchronic toxicity study of elemental Nano-Se in Sprague-Dawley rats. Life Science, 2005; 76:1989-2003.
110 Zhang JS, Wang HL, Yan XX, et al. Comparison of short-term toxicity between Nano-Se and selenite in mice. Life Science, 2005; 76:1099-1109.
111 Painter EP. The chemistry and toxicity of selenium compounds, with special reference to the selenium problem. Chemical Reviews, 1941; 28:179–213.
112 Garberg P, Stahl A, Warholm M et al. Studies of the role of DNA fragmentation in selenium toxicity. Biochem Pharmacol, 1988; 37:3401-3406.
113余日安,陈学敏,鲁文清。硒对大鼠肝细胞DNA损伤的体内体外研究。同济医科大学学报,2000; 29:212-214。
114徐辉碧,孙恩杰,杨祥良。硒的生物效应的活性氧自由基机理。华中理工大学学报,1991;19:33-37.
1 Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell, 2000; 103:239– 252.
2 Minden A, Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim. Biophys. Acta, 1997; 1333:F85–F104.
3 Lin A. Activation of the JNK signaling pathway: breaking the brake on apoptosis. Bioessays, 2003; 25:17– 24.
4 Weston C R, Davis RJ. The JNK signal transduction pathway. Curr. Opin. Genet. Dev, 2002; 12:14– 21.
5 Matsuzawa A, Nishitoh H, Tobiume K, et al. Physiological roles of ASK1-mediated signal transduction in oxidative stress-and endoplasmic reticulum stress-induced apoptosis: advanced findings from ASK1 knockout mice. Antioxid. Redox Signaling, 2002; 4:415– 425.
6 Xia Y, Makris C, Su B, et al. MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration. Proc. Natl. Acad. Sci. USA, 2000; 97:5243–5248.
7 Yujiri T, Sather S, Fanger GR, et al. Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption. Science, 1998; 282:1911– 1914.
8 Sies H. Oxidative stress: oxidants and antioxidants. Exp. Physiol 1997; 82:291– 295.
9 Droge W. Free radicals in the physiological control of cell function. Physiol. Rev, 2002; 82:47–95.
10 Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J. Cell Physiol, 2002; 192:1– 15.
11 Nathan C. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J. Clin. Invest, 2003; 111:769– 778.
12 Levonen AL, Patel RP, Brookes P, et al. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases. Antioxid. Redox Signaling, 2001; 3:215– 229.
13 Nicotera P, Bernassola F, Melino G. Nitric oxide (NO), a signaling molecule with a killer soul. Cell Death Differ. 6:931– 933; 1999. Torres, M. Mitogen -activated protein kinase pathways in redox signaling. Front. Biosci, 2003; 8:d369–d391.
14 Rhee SG, Bae, YS, Lee SR, et al. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci. STKE, 2000; 53:1– 6.
15 Mittler R, Berkowitz G. Hydrogen peroxide, a messenger with too many roles? Redox Rep, 2001; 6:69– 72.
16 Sata N, Klonowski-Stumpe H, Han B, et al. Menadione induces both necrosis and apoptosis in rat pancreatic acinar AR4-2J cells. Free Radic. Biol. Med, 1997 23:844–850.
17 Ma X, DuJ, Nakashima I, et al. Menadione biphasically controls JNK-linked cell death in leukemia Jurkat T cells. Antioxid. Redox Signaling, 2002; 4:371– 378;.
18 Ueda S, Masutani H, Nakamura H, et al. Redox control of cell death. Antioxid. Redox Signaling, 2002; 4:405– 414.
19 Ding M, Li J, Leonard SS, et al. Differential role of hydrogen peroxide in UVinduced signal transduction. Mol. Cell Biochem, 2002; 234– 235:81– 90.
20 Chan WH, Wu CC, Yu JS. Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells. J. Cell. Biochem, 2003; 90:327– 338.
21 Lee SA, Dritschilo A, Jung M. Role of ATM in oxidative stressmediated c-Jun phosphorylation in response to ionizing radiation and CdCl2. J. Biol. Chem, 2001; 276:11783–11790.
22 Yu C, Rahmani M, Dent P, Grant S. The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib. Exp. Cell Res, 2004; 295:555–566; 2004.
23 Mansat-de, M. V, Bezombes, C, Quillet-Mary, A, Bettaieb, A, D_orgeix, A. D, Laurent, G, Jaffrezou, J. P. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol. 56:867– 874; 1999.
24 Mansat-de MV, Bezombes C, Quillet-Mary A et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol, 1999 56:867– 874.
25 Shakibaei M, Schulze-Tanzil G, Takada Y, et al. Redox regulation of apoptosis by members of the TNF superfamily. Antioxid.Redox Signaling, 2005 7:482– 496.
26 Goossens V, De Vos K, Vercammen D, et al. Redox regulation of TNF signaling.Biofactors, 1999; 10:145– 156.
27 Garg AK, Aggarwal BB. Reactive oxygen intermediates in TNF signaling. Mol. Immunol, 2002;. 39:509– 517.
28 Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA, 1993; 90:7915– 7922.
29 Werner E. GTPases and reactive oxygen species: switches for killing and signaling. J. Cell Sci, 2004; 117:143– 153.
30 Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol, 2004. 4:181– 189.
31 Reinehr R, BeckerS, Eberle A, et al. Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis. J. Biol. Chem, 2005; 280:27179–27194; 2005.
32 Gu Y, Xu YC, Wu RF, et al. TNFalpha activates c-Jun amino terminal kinase through p47 (phox). Exp. Cell Res, 2002; 272:62– 74.
33 Xu YC, Wu RF, Gu Y, et al. Involvement of TRAF4 in oxidative activation of c-Jun Nterminal kinase. J. Biol. Chem, 2002; 277:28051– 28057.
34 Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90– 94; 1997.
35 Cross JV, Templeton DJ. Oxidative stress inhibits MEKK1 by sitespecific glutathionylation in the ATP-binding domain. Biochem. J, 2004; 381:675– 683.
36 Saitoh M, Nishitoh H, Fujii M, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO, 1998; J. 17:2596– 2606.
37 Nordberg J, Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med, 2001; 31:1287– 1312.
38 Gotoh Y, Cooper JA. Reactive oxygen species-and dimerizationinduced activation of apoptosis signal-regulating kinase 1 in tumor necrosis factor-alpha signal transduction. J. Biol. Chem, 1998; 273: 17477– 17482.
39 Tobiume K, Matsuzawa A, Takahashi T, et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep, 2001; 2:222– 228.
40 Liu Y, Min W. Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activityindependent manner. Circ. Res, 2002; 90:1259–1266.
41 Song JJ, Rhee JG, Suntharalingam M, et al. Role of glutaredoxin in metabolic oxidative stress: glutaredoxin as a sensor of oxidative stress mediated by H2O2. J. Biol. Chem2002; 277:46566–46575.
42 Song JJ, Lee YJ. Differential role of glutaredoxin and thioredoxin in metabolic oxidative stress-induced activation of apoptosis signalregulating kinase 1. Biochem. J, 2003; 373:845– 853.
43 Goldman EH, Chen L, Fu H. Activation of apoptosis signalregulating kinase 1 by reactive oxygen species through dephosphorylation at serine 967 and 14-3-3 dissociation. J. Biol. Chem, 2004; 279: 10442– 10449.
44 Morita K, Saitoh M, Tobiume K, et al. Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress. EMBO J, 2001; 20:6028– 6036.
45 Zhou G, Golden T, Aragon IV, et al. Ser/Thr protein hosphatase 5 inactivates hypoxia-induced activation of an apoptosis signal-regulating kinase 1/MKK-4/JNK signaling cascade. J. Biol. Chem, 2004; 279:46595– 46605.
46 Zhang W, Zheng S, Storz P, et al. Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor. J. Biol. Chem, 2005; 280:19036– 19044.
47 Yoshizumi M, Abe J, HaendelerJ, et al. Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J. Biol. Chem, 2000; 275:11706– 11712.
48 Chen K, Vita JA, Berk BC, et al. c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation. J. Biol. Chem, 2001; 276:16045– 16050.
49 Holgado-Madruga M, Wong A J. Gab1 is an integrator of cell death versus cell survival signals in oxidative stress. Mol. Cell Biol, 2003; 23:4471– 4484.
50 Sun Y, Yuan J, Liu H, Shi Z, et al. Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis. Mol. Cell Biol, 2004; 24:1531– 1539.
51 Wang T, Arifoglu P, Ronai Z, et al. Glutathione Stransferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus. J. Biol. Chem, 2001; 276:20999– 21003.
52 Adler V, Yin Z, Fuchs, SY, et al. Regulation of JNK signaling by GSTp. EMBO J, 1999; 18:1321– 1334.
53 Shen HM, Lin Y, Choksi S, et al. Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death. Mol. Cell Biol, 2004; 24:5914– 5922.
54 Boyd CS, Cadenas E. Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis. Biol. Chem, 2002; 383:411– 423.
55 Andreka P, Zang J, Dougherty C, et al. Cytoprotection by Jun kinase during nitric oxideinduced cardiac myocyte apoptosis. Circ. Res, 2001; 88:305– 312.
56 Jun CD, Pae HO, Kwak HJ, et al. Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways. Cell Immunol, 1999; 194:36– 46.
57 Nabeyrat E, Jones, G E, Fenwick PS, et al. Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells. Am. J. Physiol Lung Cell Mol. Physiol, 2003; 284:L1112–L1120.
58 Chung KC, Park J, Kim CH, et al. Tumor necrosis factoralpha and phorbol
12-myristate 13-acetate differentially modulate cytotoxic effect of nitric oxide generated by serum deprivation in neuronal PC12 cells. J. Neurochem, 1999; 72:1482–1488.
59 Go, Y. M, Patel, R. P, Maland, M. C, Park, H, Beckman, J. S, rley-Usmar, V. M, Jo, H. Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH(2)-terminal kinase. Am. J. Physiol, 1999; 277:H1647–H1653.
60 Jibiki I, Hashimoto S, Maruoka S, et al. Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates nitric oxide-induced activator protein-1 activation in human bronchial epithelial cells. Am. J. Respir. Crit. Care Med, 2003; 167:856–861.
61 Pantano C, Shrivastava P, McElhinney B, et al. Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase. J. Biol. Chem, 2003; 278:44091– 44096.
62 Shrivastava P, Pantano C, Watkin R, et al. Reactive nitrogen pecies -induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase. Mol. Cell Biol, 2004; 24:6763– 6772.
63 Park HS, Huh SH, Kim MS, et al. Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation. Proc. Natl. Acad. Sci. USA, 2000; 97:14382– 14387.
64 So HS, Park RK, Kim MS, et al. Nitric oxide inhibits c-Jun Nterminal kinase 2 (JNK2) via S-nitrosylation. Biochem. Biophys. Res. Communm 1998; 247:809–813.
65 Klatt P, Molina EP, Lamas S. Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation. J. Biol. Chem, 1999; 274:15857– 15864.
66 Park HS, Yu,JW, Cho JH, et al. Inhibition of apoptosis signal-regulating kinase 1 by nitric oxide through a thiol redox mechanism. J. Biol. Chem, 2004; 279:7584–7590.
67 Chae HJ, Kim HR, Kwak YG, et al. Signal transduction of nitric oxide donor-induced protection in hydrogen peroxide-mediated apoptosis in H9C2 cardiomyoblasts. Immunopharmacol. Immunotoxicol, 2001; 23:187– 204.
68 Carmody RJ, Cotter TG. Signalling apoptosis: a radical approach. Redox. Rep, 2001; 6:77– 90.
69 Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med, 2000; 29:323– 333.
70 Li Y, Arita Y, Koo HCet al. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am. J. Respir. Cell Mol, 2003; Biol. 29:779– 783.
71 Tournier C, Hess P, Yang DD, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science, 2000; 288:870– 874.
72 Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J. Biol. Chem, 2002; 277:10244– 10250.
73 Kharbanda S, Saxena S, Yoshida K, et al. Translocation of SAPK/JNK to mitochondria and interaction with Bcl-x(L) in response to DNA damage. J. Biol, 2000; Chem. 275:322–327.
74 Putcha GV, Le S, Frank S, et al. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron, 2003; 38:899–914.
75 Brichese L, Cazettes G, Valette A. JNK is associated with Bcl-2 and PP1 in mitochondria: paclitaxel induces its activation and its association with the phosphorylated form of Bcl-2. Cell Cycle, 1994; 3:1212– 1219.
76 Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol. Cell Biol, 1999; 19:8469– 8478.
77 Inoshita S, Takeda K, Hatai T, et al. Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress. J. Biol. Chem, 2002; 277:43730– 43734.
78 Tsuruta F, SunayamaJ, Mori Y, et al. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins. EMBO J, 2004; 23:1889– 1899.
79 Lei K,; Nimnual A, Zong WX, et al. The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol. Cell Biol, 2002; 22:4929–4942.
80 Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc. Natl. Acad. Sci. USA, 2003; 100:2432– 2437.
81 Jaattela M, Tschopp J. Caspase-independent cell death in T lymphocytes. Nat. Immunol, 2003; 4:416– 423.
82 Kroemer G, Martin. Caspase-independent cell death. Nat. Med, 2005; 11:725– 730.
83 Troyano A, Sancho P, Fernandez C, et al. The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted humanpromonocytic cells. Cell Death Differ, 2003; 10:889– 898.
84 Gardner, A. M, Xu, F. H, Fady, C, Jacoby, F. J, Duffey, D. C, Tu, Y, Lichtenstein, A. Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radic. Biol. Med, 1997; 22:73– 83.
85 Vercammen D, Beyaert R, Denecker G, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J. Exp. Med, 1998; 187:1477– 1485.
86 Vercammen D, Brouckaert G, Denecker G, et al. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.J. Exp. Med,1998; 188:919– 930.
87 Ventura JJ, Cogswell P, Flavell RA.et al. JNK potentiates TNF-stimulated necrosis by increasing the production of cytotoxic reactive oxygen species. Genes Dev, 2004; 18:2905– 2915.
88 Sakon S, Xue X, Takekawa M, et al. NFkappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. EMBO J, 2003; 22:3898– 3909.
89 Lin Y, Choksi S, Shen HM, et al. Tumor necrosis factorinduced nonapoptotic cell death requires receptor-interacting proteinmediated cellular reactive oxygen species accumulation. J. Biol. Chem, 2004; 279:10822–10828.
90 Fiers W, Beyaert R, Declercq W,et al. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18:7719– 7730; 1999.
91 Kamata H, Honda S, Maeda S, et al. Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell, 2005; 120:649– 661.
92 Conze D, Krahl T, Kennedy N, et al. c-Jun NH(2)-terminal kinase (JNK)1 and JNK2 have distinct roles in CD8(+) T cell activation. J. Exp, 2002; Med. 195:811–823.
93 Hochedlinger K, Wagner EF, Sabapathy K. Differential effects of JNK1 and JNK2 on signal specific induction of apoptosis. Oncogene, 2002; 21:2441– 2445.
94 Sabapathy K, Hochedlinger K, Nam SY, et al. Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation. Mol. Cell, 2004 15:713– 725.
95 Chung HT, Pae HO, Choi BM, et al. M. Nitric oxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun, 2001; 282:1075– 1079.
96 Vieira H, Kroemer G. Mitochondria as targets of apoptosis regulation by nitric oxide. IUBMB Life, 2003; 55:613– 616.
97 Zhou J, Schmid T, Brune B. HIF-1alpha and p53 as targets of NO in affecting cellproliferation, death and adaptation. Curr. Mol. Med, 2004 4:741– 751.
98 Kim PK, Kwon YG, Chung HT, et al. Regulation of caspases by nitric oxide. Ann. N. Y. Acad. Sci, 2002; 962:42– 52.
99 von KA, Callsen D, Brune B. NF-kappaB and AP-1 activation by nitric oxide attenuated apoptotic cell death in RAW264.7 macrophages. Mol. Biol. Cell, 1999; 10:361–372.
100 Kim YM, de Vera ME, Watkins SC, et al. Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alphainduced apoptosis by inducing heat shock protein 70 expression. J. Biol. Chem, 1997; 272:1402–1411.
101 Taimor G, Rakow A, Piper HM. Transcription activator protein 1 (AP-1) mediates NO-induced apoptosis of adult cardiomyocytes. FASEB J, 2001 15:2518– 2520.
102 Wu GS. The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol. Ther, 2004; 3:156–161.
103 Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell, 2003; 114:181– 190.
104 Liu ZG. Molecular mechanism of TNF signaling and beyond. Cell Res, 2005; 15:24–27.
105 Takeda K, Matsuzawa A, Nishitoh H, et al. Roles of MAPKKK ASK1 in stress-induced cell death. Cell Struct. Funct, 2003; 28:23– 29.
106 Lamb JA, Ventura JJ, Hess P, et al. JunD mediates survival signaling by the JNK signal transduction pathway. Mol. Cell, 2003; 11:1479–1489.
107 De SE, Zazzeroni F, Papa S, et al. Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 414:308– 313; 2001.
108 Tang G, Minemoto Y, Dibling B, et al. Inhibition of JNK activation through NF-kappaB target genes. Nature, 2001, 414:313– 317.
109 Chen F, Castranova V, Li Z, Karin M, et al. Inhibitor of nuclear factor kappaB kinase deficiency enhances oxidative stress and prolongs c-Jun NH2-terminal kinase activation induced by arsenic. Cancer Res, 2003; 63:7689– 7693.
110 Sasazuki T, Okazaki T, Tada K, Sakon-Komazawa S, et al. Genome wide analysis of TNF-inducible genes reveals that antioxidant enzymes are induced by TNF and responsible for elimination of ROS. Mol. Immunol, 2004; 41:547–551.
111 Pham CG, Bubici C, Zazzeroni F, et al. Ferritin heavy chain upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive oxygen species. Cell, 2004; 119:529– 542.
112 Djavaheri-Mergny M, Javelaud D, Wietzerbin J, et al. NFkappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels inTNFalpha-treated Ewing sarcoma cells. FEBS Lett, 2004 578:111–115.
113 Nakano H. Signaling crosstalk between NF-kappaB and JNK. Trends Immunol, 2004; 25:402–405.
114 Zhang Y, Chen F. Reactive oxygen species (ROS), troublemakers between nuclear factor-kappaB (NF-kappaB) and c-Jun NH(2)-terminal kinase (JNK). Cancer Res, 2004; 64:1902– 1905.
115 Papa S, Zazzeroni F, Pham CG, et al.. Linking JNK signaling to NF-kappaB: a key to survival. J. Cell Sci, 2004, 117:5197– 5208.
116 Papa, S, Bubici, C, Pham, C. G, Zazzeroni, F, Franzoso, G. NfkappaB meets ROS: an Firon-ic_ encounter. Cell Death Differ. 12:1259– 1262; 2005.
117 Benhar M, Dalyot I, Engelberg D, et al. Enhanced ROS production in oncogenically transformed cells potentiates c-Jun Nterminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress. Mol. Cell Biol, 2001; 21:6913– 6926.
118 Taylor BS, Alarcon LH, Billiar TR. Inducible nitric oxide synthase in the liver: regulation and function. Biochemistry (Moscow), 1998; 63:766– 781.
119 Yamaoka J, Kabashima K, Kawanishi M, et al. Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide. Biochem. Biophys. Res. Commun, 2002; 291:780– 786.
120 Song, W, Lu, X, Feng, Q. Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes. Cardiovasc. Res, 2000; 45:595– 602.
121 Binder C, Schulz M, Hiddemann W, et al. Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells. Lab. Invest, 1999; 79:1703– 1712.
122 Garban HJ, Bonavida B. Nitric oxide disrupts H2O2-dependent activation of nuclear factor kappa B: role in sensitization of human tumor cells to tumor necrosis factor-alpha-induced cytotoxicity. J. Biol.Chem, 2001; 276:8918–8923.
123 Marshall HE, Stamler JS. Nitrosative stress-induced apoptosis through inhibition of NF-kappa B. J. Biol. Chem, 2002; 277:34223– 34228.
124 Hatano E, Bennett BL, Manning AM, et al. NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha-and Fas-mediated apoptosis. Gastroenterology, 2001; 120:1251– 1262.
125 Li J, Bombeck CA, Yang S, Kim Y, et al. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J. Biol. Chem, 1999; 274:17325– 17333.
126 Bulotta S, Barsacchi R, Rotiroti D, et al. Activation of the endothelial nitric-oxide synthase by tumor necrosis factor-alpha: a novel feedback mechanism regulating cell death. J. Biol. Chem, 2001; 276:6529–6536.
127 Kim YM, Kim, TH, Chung HT, et al. Nitric oxide prevents tumor necrosis factor alphainduced rat hepatocyte apoptosis by the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8. Hepatology, 2000; 32:770– 778.
128 De NC, Sestili P, Cantoni O, et al. Nitric oxide inhibits tumor necrosis factor-alpha-induced apoptosis by reducing the generation of ceramide. Proc. Natl. Acad. Sci. USA, 2000; 97:5480– 5485.
129 Maeda S, Kamata H, Luo JL, et al. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell, 200;.121:977– 990.
2 Ip C, Lisk D J. Efficacy of cancer prevention by high-selenium garlic is primarily dependent on the action of selenium. Carcinogenesis, 1995; 16:2649–2652.
3 Clark LC, Combs GF Jr, Turnbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA, 1996; 1276:1957-1963.
4 Duffield-Lillico AJ, Reid ME, Turnbull BW, et al. Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer Epidemiol Biomarkers Prev, 2002; 11:630-6.
5 Corcoran NM, Najdovska M, Costello AJ. Inorganic selenium retards progression of experimental hormone refractory prostate cancer. J Urol, 2004; 171:907-910.
6 Zhuo HJ, Smith, AH, Steinmaus C. Selenium and lung cancer: a quantitative analysis of heterogeneity in the current epidemiological literature. Cancer Epidemiol Biomarkers Prev, 2004; 13:771-778.
7 Li L, Xie Y, El-Sayed WM, et al. Chemopreventive activity of selenocysteine prodrugs against tobacco-derived nitrosamine (NNK) induced lung tumors in the A/J mouse. J Biochem Mol Toxicol, 2005; 19:396-405.
8 El-bayoumy, K. The protective role of selenium on genetic damage and on cancer. Mutat Res, 2001, 475:123-139
9 Carson, D.A. Ribeiro, J.M. Apotosis and disease, Lancet, 1993; 341:1251-1254.
10 Stewart MS, Davis RL, Walsh LP, et al. Induction of differentiation and apoptosis by sodium selenite in human colonic carcinoma cells (HT29). Cancer Lett, 1997; 117:35-40.
11 Shen HM, Yang CF, Ong CN. Sodium selenite-induced oxidative stress and apoptosis in human hepatoma HepG2 cells. Int J Cancer, 1999; 81:820-828.
12 Jung U, Zheng X, Yoon SO, et al. Se-Methylselenocysteine induces apoptosismediated by reactive oxygen species in HL-60 cells. Free Radic Biol Med, 2001; 31:479-489.
13 Wang H Yang X Zhang Z, et al. Both calcium and ROS as common signals mediate selenite-induced apoptosis in SW480 colonic carcinoma cells. J Inorg Biochem, 2003; 97:221-230.
14 Cao TM, Hua FY, Xu CM, et al. Distinct effects of different concentrations of sodium selenite on apoptosis, cell cycle, and gene expression profile in acute promyeloytic leukemia-derived NB4 cells. Ann Hematol, 2006; 85:434-442.
15 Zhou N, Xiao H, Li TK, et al. DNA Damage-mediated Apoptosis Induced by Selenium Compounds. J Biol Chem, 2003; 278:29532-29537.
16 Zhong W, Oberley TD. Redox-mediated effects of selenium on apoptosis and cell cycle in the LNCaP human prostate cancer cell line. Cancer Res, 2001; 61:7071-7078.
17 Zuo L, Li J, Yang Y, et al. Sodium selenite induces apoptosis in acute promyelocytic leukemia-derived NB4 cells by a caspase-3-dependent mechanism and a redox pathway different from that of arsenic trioxide. Ann Hematol, 2004; 83:751-758.
18 Hu D, Liu Q, Cui H, et al. Effects of amino acids from selenium-rich silkworm pupas on human hepatoma cells. Life Sci, 2005; 77:2098-2110.
19 Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol, 1998; 10:248-253.
20 Dalton TP, Shertzer HG, Puge A. Regulation of gene expression by reactive oxygen. Pharmacol Toxicol, 1999; 39:67-101.
21 Hancock JT, Desikan R, Neill SJ. Role of reactive oxygen species in cell signalling pathways. Biochem Soc Trans, 2001; 29:345-350.
22 Kyriakis JM, Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays, 1996; 18:567-577.
23 Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med, 2000; 29:323-333.
24 Droge w. Free radicals in the physiological control of cell function. Physiol Rev, 2002; 82:47-95.
25 Torres M. Mitogen-activated protein kinase pathways in redox signaling. Front Biosci, 2003; 8:369-391.
26郭海恩,董力,余淑懿,等。硼硒联合对大鼠的急性和亚急性毒性研究。卫生研究,2002; 31(4):297-298.
27 Stacey NH, Klaassen CD. Comparison of the effects of metals on cellular injury and lipid peroxidation in isolated hepatocytes. J Toxicol environ Health, 1981; 7: 139–147.
28 Dougherty JJ, Hoekstra WG. Stimulation of lipid peroxidation in vivo by injected selenite and lack of stimulation by selenate. Proc Soc exp Biol Med, 1982; 169: 209–215.
29 Yan L, Yee JA, Boylan LM, et al. Effect of selenium compounds and thiols on human mammary tumor cells. Biol Trace Elem Res, 1991; 30:145–162.
30 Spallholz JE. On the nature of selenium toxicity and carcinostatic activity. Free Radicals Biol Med, 1994; 17:45–64.
31 Hu ML, Spallholz JE. In vitro hemolysis of rat erythrocytes by selenium compounds. Biochem Pharmacol, 1983; 32(6):957-61.
32 Hoffman JL. Selenite toxicity, depletion of liver S-adenosylmethionine, and inactivation of methionine adenosyltrasferase. Arch Biochem Biophys, 1977; 179(1):136-40.
33 Kajander EQ, Harvima RJ, Eloranta TO, et al. Metabolism, cellular actions, and cytotoxicity of selenomethionine in cultured cells. Biol Trace Elem Res, 1991; 28(1):57-68.
34 Ronai Z, Tillotson JK, Traganos F, et al. Effects of organic and inorganic selenium compounds on rat mammary tumor cells. Int J Cancer. 1995; 63(3):428-34.
35 Nano JL, Czerucka D, Menguy F, et al. Effect of selenium on the growth of three human colon cancer cell lines. Biol trance Elem Res, 1989; 20:31-43.
36 Kuchan MJ, Milner JA. Influence of intracellular glutathione on selenite-mediated growth inhibition of canine mammary tumor cells. Cancer Res, 1992; 52:1091-1095.
37 Greeder GA, Milner JA. Factors influencing the inhibitory effect of selenium on mice inoculated with Ehrlich ascites tumor cells. Science, 1980; 209:825-827.
38 Milner JA. Effect of selenium on virally induced and transplantable tumor models. Fed Proc, 1985; 44-2568-2572.
39 Medina D, Lane HW, Tracey CM. Selenium and mouse mammary tumorigenesis: aninvestigation of possible mechanisms. Cancer Res, 1983, 43:2460-2464.
40 Ip C, Lisk DJ. Efficacy of cancer prevention by high-selenium garlic is primarily dependent on the action of selenium. Carcinogenesis, 1995; 16:2649–2652.
41 Thompson HJ, Wilson A, Lu J, et al. Comparison of the effects of an organic and an inorganic form of selenium on a mammary carcinoma cell line. Carcinogenesis, 1994; 15:183-186
42 Ronai Z, Tillotson JK, Traganos F, et al. Effects of organic and inorganic selenium compounds on rat mammary tumor cells. Int J Cancer, 1995; 63:428-434.
43 Sinha R, Medina D. Inhibition of cdk2 kinase activity by methylselenocysteine in synchronized mouse mammary epithelial tumor cells. Carcinogenesis, 1997; 18:1541–1547.
44 Gopee NV, Johnson VJ, Sharma RP. Sodium selenite-induced apoptosis in murine B-lymphoma cells is associated with inhibition of protein kinase C-delta, nuclear factor kappaB, and inhibitor of apoptosis protein Toxicol Sci, 2004; 78:204-214
45张保国,杨劲松,姚乐申,沈苏南。硒酸钠对人前列腺癌细胞PC-3细胞活力和增殖的影响。中国癌症杂志,2005; 15(2):153-155.
46 Zhu, Z; Jiang W, Ganther HE, et al. Activity of Se-allylselenocysteine in the presence of methionine gamma-lyase on cell growth, DNA integrity, apoptosis, and cell-cycle regulatory molecules. Mol Carcinog, 2000, 29 (4):191–197
47 Seko Y, Saito Y, Kitahara J, et al. Active oxygen generation by the reaction of selenite with reduced glutathione in vitro. In A. Wendel (ed.), Selenium in biology and medicine. 1989; 70–73.
48 Sundaresan M, Yu ZX, Ferrans VJ, et al. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science, 1995; 270: 296-299.
49 Combs GF Jr, Gray WP. Chemopreventive agents: selenium. Pharmacol Ther, 1998; 79:179–192
50 Ganther HE. Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis, 1999; 20: 1657–1666.
51 Harrison PR, Lanfea J, Wu L, et al. Chemopreventive and growth inhibitory effects ofselenium. Biomed Environ Sci, 1997; 10, 235–245.
52 Stewart M S, Spallholz JE, Neldne, K H, et al. Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis. Free Radic Bio. Med, 1999; 26, 42–48
53朱志良,庄志雄,黄钰,等.单细胞凝胶电泳图像分析系统的研制及应用.中华劳动卫生职业病杂志, 2001; 19(4):298-300.
54 Olive PL, Banath JP, Durand RE. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the“comet”assay . Radiat Res, 1990; 122(1):86-94.
55 Tang R, Liu H, Wang T, et al. Mechanisms of selenium inhibition of cell apoptosis induced by oxysterols in rat vascular smooth muscle cells. Arch Biochem Biophys, 2005; 441(1):16-24.
56 Drake EN. Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant. Med Hypotheses, 2006; 67 (2): 318-322.
57 Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol. Today, 1994; 15:7–10.
58 Slater, AF, Nobel CS, Orrenius S. The role of intracellular oxidants in apoptosis. Biochim Biophy. Acta, 1995; 1271:59–62.
59 Kamata H, Hirata H. Redox regulation of cellular signalling. Cell Signal, 1999; 11:1–14.
60 Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol, 1999; 57:231–245.
61 Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J. Immunol Methods, 1995; 184:39-51.
62 Xing C, Peng Y, Chang R, et al. Effects of insulin-like growth factor-1 on okadaic acid-induced apoptosis in SH-SY5Y cells. Cell Biol. Int, 2005; 29:803-808.
63 Kramer GF, Ames BN. Mechanisms of mutagenicity and toxicity of sodium selenium selenite (Na2SeO3) in Salmonella typhimurium. Mutat Res, 1988; 201 (1):169-180
64 Jiang C, Hu H, Malewicz B, et al. Selenite-induced p53 Ser-15 phosphorylation andcaspase-mediated apoptosis in LNCaP human prostate cancer cells. Mol Cancer Ther. Mol Cancer Ther, 2004; 3(7):877-884.
65 Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell, 1995; 80:179-185.
66 Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med, 1996; 74:589-607.
67 Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal Kinase (JNK)-from inflammation to development. Curr Opin Cell Biol, 1998; 10:205-219.
68 Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell, 1995; 80:199-211.
69 Xia Z, Dickens M, Raingeaud, J, et al. Opposing effects of ERK and JNK-p38 MAP kinases on aopotosis. Science, 1995; 270:1326-1331.
70 Johnson NL, Gardner AM, Diner KM, et al. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. J Biol Chem, 1996; 271:3229-3237.
71 Zanke BW, Boudreau K, Rubie E, et al. The stress-activated protein kinase pathways mediate cell death following injury induced by cisplatinum, UV irradiation or heat. Curr Biol, 1996; 6:606-613.
72 Goillot E, Raingeaud J, Ranger A, et al. Mitogen-activated protein kinase -mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA, 1997; 94:3302-3307.
73 Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science, 1997; 275:90-94.
74 Rincon M, Whitmarsh A, Yang DD, et al. The JNK pathway regulates the in vivo deletion of immature CD4(+)CD8(+) thymocytes. J Exp Med, 1998; 188:1817 -1830.
75 Laszlo A. Evidence for two states of thermotolerance in mammalian cells. Int J Hyperthermia, 1988; 4(5):513-526.
76 Currie RW, Tanguay RM, Kingma JG, et al. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation, 1993; 87(3):963-971.
77 Mittler R, Berkowitz G. Hydrogen peroxide, a messenger with too many roles? Redox Rep. 2001; 6:69– 72.
78 Sata N, Klonowski-Stumpe H, Han B, et al. Menadione induces both necrosis and apoptosis in rat pancreatic acinar AR4-2J cells. Free Radic. Biol. Med, 1997; 23:844–850.
79 Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J. Cell Physiol, 2002; 192:1–15.
80 Ding M, Li J, Leonard SS, Shi X, et al. Differential role of hydrogen peroxide in UVinduced signal transduction. Mol. Cell Biochem, 2002; 234– 235:81– 90.
81 Chan WH, Wu CC, Yu JS. Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells. J. Cell. Biochem, 2003; 90:327– 338.
82 Lee SA, Dritschilo A, Jung M. Role of ATM in oxidative stressmediated c-Jun phosphorylation in response to ionizing radiation and CdCl2. J. Biol. Chem, 2001; 276:11783–11790.
83 Yu C, Rahmani M, Dent, P, Grant S. The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib. Exp. Cell Res, 2004; 295:555–566.
84 Mansat-de M V, Bezombes C, Quillet-Mary A, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol, 1999; 56:867– 874.
85 Mansat-de MV, Bezombes C, Quillet-Mary A, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol, 1999; 56:867– 874.
86 Shakibaei M, Schulze-Tanzil G, Takada Y, et al. Redox regulation of apoptosis by members of the TNF superfamily. Antioxid.Redox Signaling, 2005; 7:482– 496.
87 Goossens V, DeVos K, Vercammen D, et al. Redox regulation of TNF signaling. Biofactors, 1999; 10:145– 156.
88 Garg AK, Aggarwal BB. Reactive oxygen intermediates in TNF signaling. Mol. Immunol, 2002; 39:509– 517.
89 Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Sicence; 1997; 275:90-94.
90 Yoshizumi M, Abe J, Haendeler J, et al. Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem, 20002;
75:11706-11712.
91 Li Y, Arita Y, Koo HC, Davis JM, et al. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003; 29:779-783.
92 O’Tool D, Raisbeck M, Case JC, et al. Selenium-induced bblind staggersQ and related myths. A commentary on the extent of historical livestock losses attributed to selenosis on Wester US rangelands. Vet. Pathol, 1996; 33:104–116.
93 Oldfield JE. The two faces of selenium. J Nutr, 1987; 117:2002–2008.
94 Copper WC, Glover JR. 1974. The toxicology of selenium and its compounds. Van Nostrand Reinhold, New York, pp. 654–674.
95 Kramer G.F, Ames BN. Mechanisms of mutagenicity and toxicity of sodium selenite (Na2SeO3) in Salmonella typhimurium. Mutat Res, 1988; 201:169–180.
96 Seko Y, Imura N. Active oxygen generation as a possible mechanism of selenium toxicity, Biomed Environ Sci, 1997; 10:333–339.
97 Spallholz E. Free radical generation by selenium compounds and their prooxidant toxicity. Biomed Environ Sci, 1997; 10:260–270.
98 Spallholz JE, Palace VP, Reid TW. Methioninase and selenomethionine but not Se-methylselenocysteine generate methylselenol and superoxide in an in vitro chemiluminescent assay: implications for the nutritional carcinostatic activity of selenoamino acids. Biochem. Pharmacol, 2004; 67:547–554.
99 Terada A, Yoshida M, Seko Y, et al. Active oxygen species generation and cellular damage by additives of parenteral preparations: selenium and sulfhydryl compounds. Nutrition, 1999; 15:651–655.
100 Lu J, Kaeck M, Jiang C, et al. Thompson, Selenite induction of DNA strand breaks and apoptosis in mouse leukemic L1210 cells. Biochem Pharmacol, 1994; 47:1531–1535.
101 Ip C. Lessons from basic research in selenium and cancer prevention. Journal of Nutrition, 1998; 128:1845–1854.
102 Thompson HJ, Meeker LD, Becci PJ. Effect of combined selenium and retinyl acetate treatment on mammary carcinogenesis. Cancer Res, 1981; 41:1413–1416.
103 Temple N.J Basu TK. Selenium and cabbage and colon carcinogenesis in mice. J Natl Cancer Inst, 1987; 79:1131–1134.
104 Yan L, Yee JA, Li D, et al. Dietary supplementation of selenomethionine reduces metastasis of melanoma cells in mice. Anticancer Research, 1999; 19:1337–1342.
105 Tanaka T, Kohno H, Murakami M, et al. Suppressing effects of dietary supplementation of the organoselenium 1,4-phenylenebis(methylene)selenocyan -ate and the Citrus antioxidant auraptene on lung metastasis of melanoma cells in mice. Cancer Research, 2000; 60:3713–3716.
106 Thorlacius-Ussing O. Selenium-induced growth retardation. Histochemical and endocrinological studies on the anterior pituitaries of selenium treated rats. Danish Medical Bulletin, 1990; 37:347–358.
107 Orskov H, Flyvbjerg A. Selenium and human health. Lancet, 2000; 356:942–943.
108 Nakamuro K, Sayato Y, Tonomura M, et al. Studies on selenium related compounds. III. Acute and subacute toxicity of sodium selenate. Eisei Kagaku, 1974; 18:29–35.
109 Jia X, Li N, Chen J. A subchronic toxicity study of elemental Nano-Se in Sprague-Dawley rats. Life Science, 2005; 76:1989-2003.
110 Zhang JS, Wang HL, Yan XX, et al. Comparison of short-term toxicity between Nano-Se and selenite in mice. Life Science, 2005; 76:1099-1109.
111 Painter EP. The chemistry and toxicity of selenium compounds, with special reference to the selenium problem. Chemical Reviews, 1941; 28:179–213.
112 Garberg P, Stahl A, Warholm M et al. Studies of the role of DNA fragmentation in selenium toxicity. Biochem Pharmacol, 1988; 37:3401-3406.
113余日安,陈学敏,鲁文清。硒对大鼠肝细胞DNA损伤的体内体外研究。同济医科大学学报,2000; 29:212-214。
114徐辉碧,孙恩杰,杨祥良。硒的生物效应的活性氧自由基机理。华中理工大学学报,1991;19:33-37.
1 Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell, 2000; 103:239– 252.
2 Minden A, Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim. Biophys. Acta, 1997; 1333:F85–F104.
3 Lin A. Activation of the JNK signaling pathway: breaking the brake on apoptosis. Bioessays, 2003; 25:17– 24.
4 Weston C R, Davis RJ. The JNK signal transduction pathway. Curr. Opin. Genet. Dev, 2002; 12:14– 21.
5 Matsuzawa A, Nishitoh H, Tobiume K, et al. Physiological roles of ASK1-mediated signal transduction in oxidative stress-and endoplasmic reticulum stress-induced apoptosis: advanced findings from ASK1 knockout mice. Antioxid. Redox Signaling, 2002; 4:415– 425.
6 Xia Y, Makris C, Su B, et al. MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration. Proc. Natl. Acad. Sci. USA, 2000; 97:5243–5248.
7 Yujiri T, Sather S, Fanger GR, et al. Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption. Science, 1998; 282:1911– 1914.
8 Sies H. Oxidative stress: oxidants and antioxidants. Exp. Physiol 1997; 82:291– 295.
9 Droge W. Free radicals in the physiological control of cell function. Physiol. Rev, 2002; 82:47–95.
10 Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J. Cell Physiol, 2002; 192:1– 15.
11 Nathan C. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J. Clin. Invest, 2003; 111:769– 778.
12 Levonen AL, Patel RP, Brookes P, et al. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases. Antioxid. Redox Signaling, 2001; 3:215– 229.
13 Nicotera P, Bernassola F, Melino G. Nitric oxide (NO), a signaling molecule with a killer soul. Cell Death Differ. 6:931– 933; 1999. Torres, M. Mitogen -activated protein kinase pathways in redox signaling. Front. Biosci, 2003; 8:d369–d391.
14 Rhee SG, Bae, YS, Lee SR, et al. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci. STKE, 2000; 53:1– 6.
15 Mittler R, Berkowitz G. Hydrogen peroxide, a messenger with too many roles? Redox Rep, 2001; 6:69– 72.
16 Sata N, Klonowski-Stumpe H, Han B, et al. Menadione induces both necrosis and apoptosis in rat pancreatic acinar AR4-2J cells. Free Radic. Biol. Med, 1997 23:844–850.
17 Ma X, DuJ, Nakashima I, et al. Menadione biphasically controls JNK-linked cell death in leukemia Jurkat T cells. Antioxid. Redox Signaling, 2002; 4:371– 378;.
18 Ueda S, Masutani H, Nakamura H, et al. Redox control of cell death. Antioxid. Redox Signaling, 2002; 4:405– 414.
19 Ding M, Li J, Leonard SS, et al. Differential role of hydrogen peroxide in UVinduced signal transduction. Mol. Cell Biochem, 2002; 234– 235:81– 90.
20 Chan WH, Wu CC, Yu JS. Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells. J. Cell. Biochem, 2003; 90:327– 338.
21 Lee SA, Dritschilo A, Jung M. Role of ATM in oxidative stressmediated c-Jun phosphorylation in response to ionizing radiation and CdCl2. J. Biol. Chem, 2001; 276:11783–11790.
22 Yu C, Rahmani M, Dent P, Grant S. The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib. Exp. Cell Res, 2004; 295:555–566; 2004.
23 Mansat-de, M. V, Bezombes, C, Quillet-Mary, A, Bettaieb, A, D_orgeix, A. D, Laurent, G, Jaffrezou, J. P. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol. 56:867– 874; 1999.
24 Mansat-de MV, Bezombes C, Quillet-Mary A et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol, 1999 56:867– 874.
25 Shakibaei M, Schulze-Tanzil G, Takada Y, et al. Redox regulation of apoptosis by members of the TNF superfamily. Antioxid.Redox Signaling, 2005 7:482– 496.
26 Goossens V, De Vos K, Vercammen D, et al. Redox regulation of TNF signaling.Biofactors, 1999; 10:145– 156.
27 Garg AK, Aggarwal BB. Reactive oxygen intermediates in TNF signaling. Mol. Immunol, 2002;. 39:509– 517.
28 Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA, 1993; 90:7915– 7922.
29 Werner E. GTPases and reactive oxygen species: switches for killing and signaling. J. Cell Sci, 2004; 117:143– 153.
30 Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol, 2004. 4:181– 189.
31 Reinehr R, BeckerS, Eberle A, et al. Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis. J. Biol. Chem, 2005; 280:27179–27194; 2005.
32 Gu Y, Xu YC, Wu RF, et al. TNFalpha activates c-Jun amino terminal kinase through p47 (phox). Exp. Cell Res, 2002; 272:62– 74.
33 Xu YC, Wu RF, Gu Y, et al. Involvement of TRAF4 in oxidative activation of c-Jun Nterminal kinase. J. Biol. Chem, 2002; 277:28051– 28057.
34 Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90– 94; 1997.
35 Cross JV, Templeton DJ. Oxidative stress inhibits MEKK1 by sitespecific glutathionylation in the ATP-binding domain. Biochem. J, 2004; 381:675– 683.
36 Saitoh M, Nishitoh H, Fujii M, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO, 1998; J. 17:2596– 2606.
37 Nordberg J, Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med, 2001; 31:1287– 1312.
38 Gotoh Y, Cooper JA. Reactive oxygen species-and dimerizationinduced activation of apoptosis signal-regulating kinase 1 in tumor necrosis factor-alpha signal transduction. J. Biol. Chem, 1998; 273: 17477– 17482.
39 Tobiume K, Matsuzawa A, Takahashi T, et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep, 2001; 2:222– 228.
40 Liu Y, Min W. Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activityindependent manner. Circ. Res, 2002; 90:1259–1266.
41 Song JJ, Rhee JG, Suntharalingam M, et al. Role of glutaredoxin in metabolic oxidative stress: glutaredoxin as a sensor of oxidative stress mediated by H2O2. J. Biol. Chem2002; 277:46566–46575.
42 Song JJ, Lee YJ. Differential role of glutaredoxin and thioredoxin in metabolic oxidative stress-induced activation of apoptosis signalregulating kinase 1. Biochem. J, 2003; 373:845– 853.
43 Goldman EH, Chen L, Fu H. Activation of apoptosis signalregulating kinase 1 by reactive oxygen species through dephosphorylation at serine 967 and 14-3-3 dissociation. J. Biol. Chem, 2004; 279: 10442– 10449.
44 Morita K, Saitoh M, Tobiume K, et al. Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress. EMBO J, 2001; 20:6028– 6036.
45 Zhou G, Golden T, Aragon IV, et al. Ser/Thr protein hosphatase 5 inactivates hypoxia-induced activation of an apoptosis signal-regulating kinase 1/MKK-4/JNK signaling cascade. J. Biol. Chem, 2004; 279:46595– 46605.
46 Zhang W, Zheng S, Storz P, et al. Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor. J. Biol. Chem, 2005; 280:19036– 19044.
47 Yoshizumi M, Abe J, HaendelerJ, et al. Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J. Biol. Chem, 2000; 275:11706– 11712.
48 Chen K, Vita JA, Berk BC, et al. c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation. J. Biol. Chem, 2001; 276:16045– 16050.
49 Holgado-Madruga M, Wong A J. Gab1 is an integrator of cell death versus cell survival signals in oxidative stress. Mol. Cell Biol, 2003; 23:4471– 4484.
50 Sun Y, Yuan J, Liu H, Shi Z, et al. Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis. Mol. Cell Biol, 2004; 24:1531– 1539.
51 Wang T, Arifoglu P, Ronai Z, et al. Glutathione Stransferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus. J. Biol. Chem, 2001; 276:20999– 21003.
52 Adler V, Yin Z, Fuchs, SY, et al. Regulation of JNK signaling by GSTp. EMBO J, 1999; 18:1321– 1334.
53 Shen HM, Lin Y, Choksi S, et al. Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death. Mol. Cell Biol, 2004; 24:5914– 5922.
54 Boyd CS, Cadenas E. Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis. Biol. Chem, 2002; 383:411– 423.
55 Andreka P, Zang J, Dougherty C, et al. Cytoprotection by Jun kinase during nitric oxideinduced cardiac myocyte apoptosis. Circ. Res, 2001; 88:305– 312.
56 Jun CD, Pae HO, Kwak HJ, et al. Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways. Cell Immunol, 1999; 194:36– 46.
57 Nabeyrat E, Jones, G E, Fenwick PS, et al. Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells. Am. J. Physiol Lung Cell Mol. Physiol, 2003; 284:L1112–L1120.
58 Chung KC, Park J, Kim CH, et al. Tumor necrosis factoralpha and phorbol
12-myristate 13-acetate differentially modulate cytotoxic effect of nitric oxide generated by serum deprivation in neuronal PC12 cells. J. Neurochem, 1999; 72:1482–1488.
59 Go, Y. M, Patel, R. P, Maland, M. C, Park, H, Beckman, J. S, rley-Usmar, V. M, Jo, H. Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH(2)-terminal kinase. Am. J. Physiol, 1999; 277:H1647–H1653.
60 Jibiki I, Hashimoto S, Maruoka S, et al. Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates nitric oxide-induced activator protein-1 activation in human bronchial epithelial cells. Am. J. Respir. Crit. Care Med, 2003; 167:856–861.
61 Pantano C, Shrivastava P, McElhinney B, et al. Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase. J. Biol. Chem, 2003; 278:44091– 44096.
62 Shrivastava P, Pantano C, Watkin R, et al. Reactive nitrogen pecies -induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase. Mol. Cell Biol, 2004; 24:6763– 6772.
63 Park HS, Huh SH, Kim MS, et al. Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation. Proc. Natl. Acad. Sci. USA, 2000; 97:14382– 14387.
64 So HS, Park RK, Kim MS, et al. Nitric oxide inhibits c-Jun Nterminal kinase 2 (JNK2) via S-nitrosylation. Biochem. Biophys. Res. Communm 1998; 247:809–813.
65 Klatt P, Molina EP, Lamas S. Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation. J. Biol. Chem, 1999; 274:15857– 15864.
66 Park HS, Yu,JW, Cho JH, et al. Inhibition of apoptosis signal-regulating kinase 1 by nitric oxide through a thiol redox mechanism. J. Biol. Chem, 2004; 279:7584–7590.
67 Chae HJ, Kim HR, Kwak YG, et al. Signal transduction of nitric oxide donor-induced protection in hydrogen peroxide-mediated apoptosis in H9C2 cardiomyoblasts. Immunopharmacol. Immunotoxicol, 2001; 23:187– 204.
68 Carmody RJ, Cotter TG. Signalling apoptosis: a radical approach. Redox. Rep, 2001; 6:77– 90.
69 Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med, 2000; 29:323– 333.
70 Li Y, Arita Y, Koo HCet al. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am. J. Respir. Cell Mol, 2003; Biol. 29:779– 783.
71 Tournier C, Hess P, Yang DD, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science, 2000; 288:870– 874.
72 Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J. Biol. Chem, 2002; 277:10244– 10250.
73 Kharbanda S, Saxena S, Yoshida K, et al. Translocation of SAPK/JNK to mitochondria and interaction with Bcl-x(L) in response to DNA damage. J. Biol, 2000; Chem. 275:322–327.
74 Putcha GV, Le S, Frank S, et al. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron, 2003; 38:899–914.
75 Brichese L, Cazettes G, Valette A. JNK is associated with Bcl-2 and PP1 in mitochondria: paclitaxel induces its activation and its association with the phosphorylated form of Bcl-2. Cell Cycle, 1994; 3:1212– 1219.
76 Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol. Cell Biol, 1999; 19:8469– 8478.
77 Inoshita S, Takeda K, Hatai T, et al. Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress. J. Biol. Chem, 2002; 277:43730– 43734.
78 Tsuruta F, SunayamaJ, Mori Y, et al. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins. EMBO J, 2004; 23:1889– 1899.
79 Lei K,; Nimnual A, Zong WX, et al. The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol. Cell Biol, 2002; 22:4929–4942.
80 Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc. Natl. Acad. Sci. USA, 2003; 100:2432– 2437.
81 Jaattela M, Tschopp J. Caspase-independent cell death in T lymphocytes. Nat. Immunol, 2003; 4:416– 423.
82 Kroemer G, Martin. Caspase-independent cell death. Nat. Med, 2005; 11:725– 730.
83 Troyano A, Sancho P, Fernandez C, et al. The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted humanpromonocytic cells. Cell Death Differ, 2003; 10:889– 898.
84 Gardner, A. M, Xu, F. H, Fady, C, Jacoby, F. J, Duffey, D. C, Tu, Y, Lichtenstein, A. Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radic. Biol. Med, 1997; 22:73– 83.
85 Vercammen D, Beyaert R, Denecker G, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J. Exp. Med, 1998; 187:1477– 1485.
86 Vercammen D, Brouckaert G, Denecker G, et al. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.J. Exp. Med,1998; 188:919– 930.
87 Ventura JJ, Cogswell P, Flavell RA.et al. JNK potentiates TNF-stimulated necrosis by increasing the production of cytotoxic reactive oxygen species. Genes Dev, 2004; 18:2905– 2915.
88 Sakon S, Xue X, Takekawa M, et al. NFkappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. EMBO J, 2003; 22:3898– 3909.
89 Lin Y, Choksi S, Shen HM, et al. Tumor necrosis factorinduced nonapoptotic cell death requires receptor-interacting proteinmediated cellular reactive oxygen species accumulation. J. Biol. Chem, 2004; 279:10822–10828.
90 Fiers W, Beyaert R, Declercq W,et al. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18:7719– 7730; 1999.
91 Kamata H, Honda S, Maeda S, et al. Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell, 2005; 120:649– 661.
92 Conze D, Krahl T, Kennedy N, et al. c-Jun NH(2)-terminal kinase (JNK)1 and JNK2 have distinct roles in CD8(+) T cell activation. J. Exp, 2002; Med. 195:811–823.
93 Hochedlinger K, Wagner EF, Sabapathy K. Differential effects of JNK1 and JNK2 on signal specific induction of apoptosis. Oncogene, 2002; 21:2441– 2445.
94 Sabapathy K, Hochedlinger K, Nam SY, et al. Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation. Mol. Cell, 2004 15:713– 725.
95 Chung HT, Pae HO, Choi BM, et al. M. Nitric oxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun, 2001; 282:1075– 1079.
96 Vieira H, Kroemer G. Mitochondria as targets of apoptosis regulation by nitric oxide. IUBMB Life, 2003; 55:613– 616.
97 Zhou J, Schmid T, Brune B. HIF-1alpha and p53 as targets of NO in affecting cellproliferation, death and adaptation. Curr. Mol. Med, 2004 4:741– 751.
98 Kim PK, Kwon YG, Chung HT, et al. Regulation of caspases by nitric oxide. Ann. N. Y. Acad. Sci, 2002; 962:42– 52.
99 von KA, Callsen D, Brune B. NF-kappaB and AP-1 activation by nitric oxide attenuated apoptotic cell death in RAW264.7 macrophages. Mol. Biol. Cell, 1999; 10:361–372.
100 Kim YM, de Vera ME, Watkins SC, et al. Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alphainduced apoptosis by inducing heat shock protein 70 expression. J. Biol. Chem, 1997; 272:1402–1411.
101 Taimor G, Rakow A, Piper HM. Transcription activator protein 1 (AP-1) mediates NO-induced apoptosis of adult cardiomyocytes. FASEB J, 2001 15:2518– 2520.
102 Wu GS. The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol. Ther, 2004; 3:156–161.
103 Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell, 2003; 114:181– 190.
104 Liu ZG. Molecular mechanism of TNF signaling and beyond. Cell Res, 2005; 15:24–27.
105 Takeda K, Matsuzawa A, Nishitoh H, et al. Roles of MAPKKK ASK1 in stress-induced cell death. Cell Struct. Funct, 2003; 28:23– 29.
106 Lamb JA, Ventura JJ, Hess P, et al. JunD mediates survival signaling by the JNK signal transduction pathway. Mol. Cell, 2003; 11:1479–1489.
107 De SE, Zazzeroni F, Papa S, et al. Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 414:308– 313; 2001.
108 Tang G, Minemoto Y, Dibling B, et al. Inhibition of JNK activation through NF-kappaB target genes. Nature, 2001, 414:313– 317.
109 Chen F, Castranova V, Li Z, Karin M, et al. Inhibitor of nuclear factor kappaB kinase deficiency enhances oxidative stress and prolongs c-Jun NH2-terminal kinase activation induced by arsenic. Cancer Res, 2003; 63:7689– 7693.
110 Sasazuki T, Okazaki T, Tada K, Sakon-Komazawa S, et al. Genome wide analysis of TNF-inducible genes reveals that antioxidant enzymes are induced by TNF and responsible for elimination of ROS. Mol. Immunol, 2004; 41:547–551.
111 Pham CG, Bubici C, Zazzeroni F, et al. Ferritin heavy chain upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive oxygen species. Cell, 2004; 119:529– 542.
112 Djavaheri-Mergny M, Javelaud D, Wietzerbin J, et al. NFkappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels inTNFalpha-treated Ewing sarcoma cells. FEBS Lett, 2004 578:111–115.
113 Nakano H. Signaling crosstalk between NF-kappaB and JNK. Trends Immunol, 2004; 25:402–405.
114 Zhang Y, Chen F. Reactive oxygen species (ROS), troublemakers between nuclear factor-kappaB (NF-kappaB) and c-Jun NH(2)-terminal kinase (JNK). Cancer Res, 2004; 64:1902– 1905.
115 Papa S, Zazzeroni F, Pham CG, et al.. Linking JNK signaling to NF-kappaB: a key to survival. J. Cell Sci, 2004, 117:5197– 5208.
116 Papa, S, Bubici, C, Pham, C. G, Zazzeroni, F, Franzoso, G. NfkappaB meets ROS: an Firon-ic_ encounter. Cell Death Differ. 12:1259– 1262; 2005.
117 Benhar M, Dalyot I, Engelberg D, et al. Enhanced ROS production in oncogenically transformed cells potentiates c-Jun Nterminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress. Mol. Cell Biol, 2001; 21:6913– 6926.
118 Taylor BS, Alarcon LH, Billiar TR. Inducible nitric oxide synthase in the liver: regulation and function. Biochemistry (Moscow), 1998; 63:766– 781.
119 Yamaoka J, Kabashima K, Kawanishi M, et al. Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide. Biochem. Biophys. Res. Commun, 2002; 291:780– 786.
120 Song, W, Lu, X, Feng, Q. Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes. Cardiovasc. Res, 2000; 45:595– 602.
121 Binder C, Schulz M, Hiddemann W, et al. Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells. Lab. Invest, 1999; 79:1703– 1712.
122 Garban HJ, Bonavida B. Nitric oxide disrupts H2O2-dependent activation of nuclear factor kappa B: role in sensitization of human tumor cells to tumor necrosis factor-alpha-induced cytotoxicity. J. Biol.Chem, 2001; 276:8918–8923.
123 Marshall HE, Stamler JS. Nitrosative stress-induced apoptosis through inhibition of NF-kappa B. J. Biol. Chem, 2002; 277:34223– 34228.
124 Hatano E, Bennett BL, Manning AM, et al. NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha-and Fas-mediated apoptosis. Gastroenterology, 2001; 120:1251– 1262.
125 Li J, Bombeck CA, Yang S, Kim Y, et al. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J. Biol. Chem, 1999; 274:17325– 17333.
126 Bulotta S, Barsacchi R, Rotiroti D, et al. Activation of the endothelial nitric-oxide synthase by tumor necrosis factor-alpha: a novel feedback mechanism regulating cell death. J. Biol. Chem, 2001; 276:6529–6536.
127 Kim YM, Kim, TH, Chung HT, et al. Nitric oxide prevents tumor necrosis factor alphainduced rat hepatocyte apoptosis by the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8. Hepatology, 2000; 32:770– 778.
128 De NC, Sestili P, Cantoni O, et al. Nitric oxide inhibits tumor necrosis factor-alpha-induced apoptosis by reducing the generation of ceramide. Proc. Natl. Acad. Sci. USA, 2000; 97:5480– 5485.
129 Maeda S, Kamata H, Luo JL, et al. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell, 200;.121:977– 990.