Cr（Ⅵ）诱导肝细胞线粒体呼吸链功能紊乱在细胞凋亡与早衰中的分子机制
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摘要
研究背景
铬及其化合物已成为严重威胁人类健康的不可忽视的环境污染物,广泛应用于工业生产过程如冶金、电镀等。铬的职业暴露可引发的健康危害包括接触性皮炎、鼻中隔穿孔以及支气管癌等。Cr(Ⅵ)可引发急慢性肝炎以及肝损伤。Cr(Ⅵ)最主要的细胞毒作用是诱导凋亡的发生,虽然已有大量的研究证实ROS在Cr(Ⅵ)诱导的细胞毒性中发挥极为重要的作用,但Cr(Ⅵ)具体的作用机制仍不十分清楚,比如ROS如何产生的,Cr(Ⅵ)如何诱导p53、NF-kB的激活等等。本研究除了阐明Cr(Ⅵ)诱导肝细胞凋亡的作用机制外,也对其诱导肝细胞发生早衰进行了探讨。细胞早衰是近年来的研究热点,是-种不同于凋亡的细胞毒性结局。环境中Cr(Ⅵ)的人群暴露多为低剂量长期的暴露,故研究早衰具有极其重要的实际意义。早衰被认为是抑制细胞恶性转化的一道有力屏障,而凋亡是细胞的程序性死亡,故本研究对Cr(Ⅵ)诱导早衰与凋亡的同时探讨将对阐明Cr(Ⅵ)的致癌机制提供一定的实验依据。
研究方法
1.Cr(Ⅵ)诱导肝细胞凋亡
首先用MTT法检测不同浓度的Cr(Ⅵ)(0-512μM)对L-02肝细胞存活率的影响,选取适宜的浓度(存活率>70%)进行后续试验。DNA-Ladder法检测Cr(Ⅵ)暴露后DNA的损伤；末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)法对完整的单个凋亡细胞核或凋亡小体进行原位染色,反应细胞凋亡；Annexin V-FITC/PI双染法借助流式细胞仪检测Cr(Ⅵ)暴露后的早期和晚期凋亡细胞；用western blotting法检测不同浓度的Cr(Ⅵ)处理后对PARP、 Caspase-8、Caspase-9、Caspase-3蛋白表达水平的影响,进而分析细胞凋亡通路；比色法检测Cr(Ⅵ)对Caspase-3活性的影响；通过对热休克蛋白HSP70与HSP90表达水平的分析研究HSP与caspase-3的关系；通过检测GSH、MDA以及SOD研究Cr(Ⅵ)对细胞内抗氧化系统的影响以及产生自由基的能力。
2.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱
Cr(Ⅵ)处理后提取肝细胞线粒体用Clark氧电极通过记录氧耗(nmol/min/mg protein)来检测线粒体呼吸功能,指标包括三态呼吸、四态呼吸以及呼吸控制率等；用不同浓度的Cr(Ⅵ)(0、4、8、16、32μM)处理L-02肝细胞24h。提取线粒体用试剂盒检测线粒体呼吸链复合体I-V的活性；提取RNA并经逆转录成DNA后用real-timePCR法检测复合体I-VmRNA基因的表达；本研究同时用western blotting法检测了Cr(Ⅵ)对线粒体呼吸链复合体I-V蛋白表达水平的影响；同时检测了Cr(Ⅵ)对线粒体膜电位(△Ψm)、PTP孔开放度、膜通透性以及对肝细胞中LDH,ALT,AST活性的影响；高效液相色谱法检测Cr(Ⅵ)对肝细胞内腺苷酸含量(ATP、ADP、AMP)、ATP/ADP比值、以及能荷(EC)的影响。
3.Cr(Ⅵ)诱导的肝细胞线粒体呼吸链功能紊乱在凋亡中的作用
用PBS以及16、32μM Cr(Ⅵ)处理L-02肝细胞24h,经荧光探针CM-H2DCFDA孵育后在荧光显微镜下检测ROS的生成情况,同时用已被证实可诱导ROS生成增加MRCC I抑制剂ROT用做阳性对照；用不同MRCC的底物作用于肝细胞线粒体后测定Cr(Ⅵ)还原速率检测Cr(Ⅵ)在线粒体中的还原位点；本研究推断ROS在Cr(Ⅵ)诱导的L-02肝细胞毒性中发挥了重要的作用,故用抗氧化剂NAC抑制ROS来证实；检测NAC对不同浓度Cr(Ⅵ)暴露后的肝细胞ATP含量的影响；检测Cr(Ⅵ)暴露后对p53mRNA以及蛋白表达水平的影响以及NAC的作用；检测NAC预处理对不同浓度的Cr(Ⅵ)处理后热休克蛋白表达的影响；流式细胞仪检测肝细胞细胞周期的分布情况；为阐明Cr(Ⅵ)诱导的L-02肝细胞细胞周期分布变化的原因,本研究用荧光定量PCR法以及western blotting法检测了Cr(Ⅵ)作用后细胞S期检查点蛋白(Tof1、Mrc1)以及S期相关基因(CDK2,Cyclin E)的表达水平,同时也检测了G2/M期检查点蛋白(BubR1、Mad2)以及相关基因(Cyclin B、CDC25)表达水平的影响；未验证Cr(Ⅵ)诱导的细胞周期阻滞是否与p53相关,本研究选用特异性的p53抑制剂PFT-a来阻断p53的作用；检测PFT-a对不同浓度Cr(Ⅵ)处理组L-02肝细胞中p53、Mrc1、BubR1以及细胞周期分布的影响；除p53外,本研究也检测了Cr(Ⅵ)对NF-κB、PI3K/AKT通路的影响以及ROS在其中的作用。
4.Cr(Ⅵ)诱导的肝细胞线粒体呼吸链功能紊乱在早衰中的作用
用PBS或10nM的Cr(Ⅵ)处理L-02肝细胞,每周2次,每次24h,持续4周；SA-B-Gal染色检测早衰的发生；流式细胞仪细胞周期分布的分析；检测Cr(Ⅵ)对早衰L-02肝细胞中衰老、坏死、凋亡以及增殖细胞比例的影响,从而鉴别早衰与凋亡；用氧化敏感型荧光探针CM-H2DCFDA在荧光显微镜下检测ROS的生成情况；由于线粒体呼吸链复合体的抑制通常引发线粒体超氧化物的产生,本研究用线粒体超氧化物探针MitoSOX Red特异性标记超氧化物的产生情况；检测Cr(Ⅵ)诱导的早衰L-02肝细胞中线粒体呼吸链复合体(MRCC) Ⅰ-Ⅴ活性；检测有或无NAC预处理作用时Cr(Ⅵ)对肝细胞中p53mRNA表达水平的影响；Cr(Ⅵ)诱导的早衰L-02肝细胞中p53磷酸化蛋白的检测；有或无NAC预处理作用时Cr(Ⅵ)对肝细胞中衰老通路(p53-p21WAF1/CIP1、Rb-p16INK4a)蛋白水平以及对肝细胞中促存活基因以及细胞周期S期相关蛋白表达水平的影响；为证实是否ROS诱导细胞发生早衰的功能完全依赖于p53的功能,本研究用脂质体转染在肝细胞中敲除p53,用无靶基因作用的Scramble(Scr)做为对照；用PBS或10nM的Cr(Ⅵ)处理L-02-Scr以及L-02-p53shRNA,每周2次,每次24h,持续4周；检测p53敲除对Cr(Ⅵ)诱导L-02肝细胞早衰发生以及检测衰老细胞百分比的影响；检测p53敲除对Cr(Ⅵ)暴露后肝细胞中促存活基因以及细胞周期S期相关蛋白表达水平的影响。
研究结果
1.Cr(Ⅵ)诱导肝细胞凋亡
Cr(Ⅵ)对L-02肝细胞存活率的抑制存在明显的剂量-效应依存关系,为保证细胞存活率在一个比较适宜的范围(>70%)以及考虑到统计学分析的需要,本研究各个指标检测选用Cr(Ⅵ)的处理浓度为4、8、16、32μM。Cr(Ⅵ)处理后的肝细胞呈现凋亡细胞特征性的梯状DNA条带。TUNEL实验证实Cr(Ⅵ)处理后损伤的DNA片段增加,即Cr(Ⅵ)可以诱导L-02肝细胞发生凋亡,且凋亡的细胞数目随着Cr(Ⅵ)染毒浓度的增加而增加。Cr(Ⅵ)处理后无论是凋亡细胞或是晚期凋亡坏死细胞都随Cr(Ⅵ)染毒浓度的增加而增加,呈现明显剂量-效应关系。Caspase-8参与的死亡受体依赖性凋亡通路以及Caspase-9参与的非死亡受体依赖性凋亡通路均参与到了Cr(Ⅵ)诱导的肝细胞凋亡之中。Cr(Ⅵ)通过抑制HSP70与HSP90的表达水平而影响caspase-3的活性。对GSH、MDA以及SOD的检测结果说明Cr(Ⅵ)明显影响细胞内抗氧化系统以及自由基产生能力。
2.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱
Cr(Ⅵ)抑制线粒体三态呼吸而并未对四态呼吸产生影响,呼吸控制率RCR明显降低。不同浓度的Cr(Ⅵ)处理显著抑制了MRCCⅠ和Ⅱ的活性、mRNA以及蛋白表达水平,尤其是MRCCⅠ。Cr(Ⅵ)处理后,线粒体的膜电位降低、PTP开放度增大、膜的通透性升高,酶类外溢。Cr(Ⅵ)降低肝细胞内腺苷酸含量、ATP/ADP比值以及EC。
3.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱在凋亡中的作用
Cr(Ⅵ)处理组肝细胞ROS生成量随着染毒浓度的增加而增加。用MRCCⅠ的底物谷氨酸以及苹果酸(Glu+Mal)处理肝细胞线粒体时Cr(Ⅵ)的还原速率均明显增大,说明Cr(Ⅵ)在MRCCⅠ处被还原。NAC阻断了各个Cr(Ⅵ)处理组中ROS的堆积,肝细胞中ATP降低趋势有明显地缓解,抑制细胞凋亡通路的激活从而抑制了肝细胞凋亡的发生。Cr(Ⅵ)处理组细胞p53蛋白表达水平明显增加,且呈现明显的剂量-效应关系,而NAC明显抑制了p53mRNA水平的表达。Cr(Ⅵ)是通过调节ROS进而调节热休克蛋白的表达而激活caspase-3。低浓度Cr(Ⅵ)(4μM)处理组细胞发生明显的S期细胞周期阻滞,而高浓度Cr(Ⅵ)(16、32gM)处理组细胞发生明显的G2/M期细胞周期阻滞。进一步研究发现低浓度Cr(Ⅵ)导致L-02肝细胞发生S期细胞阻滞的原因可能为诱导S期检查点蛋白基因(Tof1、Mrc1)以及S期相关基因(CDK2、Cyclin E)表达水平下降有关；而高浓度Cr(Ⅵ)导致L-02肝细胞发生G2/M期细胞阻滞的原因可能为诱导G2/M期检查点蛋白基因(BubR1、Mad2)以及S期相关基因(Cyclin B、CDC25)表达水平下降有关。NAC以及PFT-a预处理明显缓解了细胞的S期或G2/M期阻滞,说明Cr(Ⅵ)通过调控ROS水平进而调控p53而诱导L-02肝细胞细胞周期阻滞。Cr(Ⅵ)诱导的ROS堆积除作用于p53外也作用于NF-kB与PI3K/AKT通路,参与细胞毒作用。
4.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱在早衰中的作用
低剂量长期Cr(Ⅵ)暴露引发L-02肝细胞发生早衰。早衰细胞出现明显的S期细胞周期阻滞,SA-β-Gal活性明显增高。与Cr(Ⅵ)诱导肝细胞凋亡中ROS的生成机制类似,早衰细胞中Cr(Ⅵ)可以选择性作用于线粒体呼吸链的易感位点MRCCⅠ和MRCC Ⅱ而诱导ROS的大量堆积。衰老细胞中磷酸化p53(ser15)明显升高,p53被明显激活, NAC预处理明显降低了对照组和早衰细胞中p53水平,提示ROS可以对p53进行转录调控。对Cr(Ⅵ)诱导的早衰肝细胞中衰老通路的分析发现p53-p21WAF1/CIP1通路,而不是Rb-p16INK4a通路参与了肝细胞早衰的过程。早衰细胞中ROS调节p53后进而调节促存活基因Bcl-2、Mcl-1的表达而抑制肝细胞的增殖能力,通过调节S期相关蛋白CDK2、Cyclin E进而诱导S期细胞周期阻滞。NAC预处理明显抑制了细胞早衰的发生。肝细胞中敲除p53后发现p53的缺失抑制了细胞早衰的发生。虽然Cr(Ⅵ)处理后的L-02-p53shRNA细胞未发生早衰,但其ROS生成量仍明显增高,证实Cr(Ⅵ)诱导的L-02细胞发生早衰依赖于ROS,而ROS功能的发挥完全依赖于p53。
研究结论
1.Cr(Ⅵ)诱导L-02肝细胞发生凋亡,且Caspase-8介导的死亡受体依赖性凋亡通路以及Caspase-9介导的非死亡受体依赖性凋亡通路均参与其中。Caspase-3的活化与热休克蛋白(HSP70,HSP90)的抑制有关。
2.Cr(Ⅵ)诱导肝细胞线粒体呼吸链的抑制以及能量代谢障碍,表现为三态呼吸的抑制、呼吸控制率的降低和ATP的耗竭等。
3.线粒体呼吸链功能紊乱的直接结果是ROS的大量堆积。Cr(Ⅵ)通过抑制线粒体呼吸链复合体Ⅰ、Ⅱ的活性而诱导ROS水平剧增,且Cr(Ⅵ)在MRCC Ⅰ处被还原。低浓度或高浓度Cr(Ⅵ)处理分别发生S期或G2/M期细胞周期阻滞,其机制与Cr(Ⅵ)作用后ROS调控p53,后者进一步调控细胞周期检查点蛋白以及细胞周期相关蛋白有关。Cr(Ⅵ)诱导的细胞凋亡依赖于ROS-p53、NF-κB以及PI3K/AKT通路。4.低剂量长期Cr(Ⅵ)暴露引发L-02肝细胞发生早衰,以不可逆性的S期细胞周期阻滞以及SA-β-Gal活性的明显增高为特征。p53-p21WAF1/CIP1通路参与早衰过程。ROS通过调节促存活基因以及S期相关蛋白而诱导S期周期阻滞。敲除p53的肝细胞经Cr(Ⅵ)诱导后不发生早衰,提示早衰的发生完全依赖于ROS-p53功能。
Background
Chromium and its compounds are non-negligible pollutant that have became a serious threat to public health in the world with increasing use in diverse industrial processes, including metallurgy, electroplating, leather tanning and chroming. Occupational exposure to chromium is associated with several adverse effects of health, such as contact dermatitis, nasal perforation, and bronchiogenic cancer. Cr(Ⅵ) can induce toxic and chronic hepatitis and severe liver damage. The main cytotoxicity of Cr(Ⅵ) is apoptosis induction. Although it is believed that ROS plays an important role in the toxicity of Cr(Ⅵ), the related mechanisms still remain unclear, for example, how and where ROS are produced, how does Cr(Ⅵ) induce the activation of p53and NF-kB? In addition to explore the mechanisms for Cr(Ⅵ)-induced apoptosis, we also placed high emphasis on Cr(Ⅵ)-induce premature senescence. Premature senescence, which has been studied a lot recently, is totally different from apoptosis. The occupational exposure to Cr(Ⅵ) is low dose and long term, that is why the research on Cr(Ⅵ)-induced premature senescence has great significance. Premature senescence can be also viewed as an intrinsic cellular barrier against tumorigenesis, while apoptosis is programmed cell death. The present research which focused on both the apoptisis and premature senescence, has depicted an original cellular toxic phenomenon in response to Cr(Ⅵ) that may constitute a protective mechanism to the tumorigenic effect of chromium in humans.
Methods
1. Cr(Ⅵ) induces apoptosis in L-02hepatocytes
Using MTT method to detect the effect of different doses of Cr(Ⅵ)(0-512μM) exposure on L-02hepatocytes survival rate, and choose proper Cr(Ⅵ) treated concentrations (survival rate>70%) for the following experiments. Cr(Ⅵ)-induced DNA damage was examined by DNA-Ladder method. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used to detect the occurrence of apoptosis in the cells. Annexin V-FITC/PI double stain was used to detect the apoptotic cells in both the early stage and late stage. Western blotting was processed in the study to detect the effect of Cr(Ⅵ) on the expression levels of PARP, Caspase-8, Caspase-9and Caspase-3to further analyze the apoptic pathway. The activity of Caspase-3was also measured using colorimetry. By analyzing the expression levels of heat shock proteins HSP70and HSP90after Cr(Ⅵ) exposure to explore the correlation between HSP and caspase-3.By detecting the levels of GSH, MDA and SOD to study the effect of Cr(Ⅵ) on cellular antioxidant system and the capability of the hepatocytes to produce oxygen radicals.
2Cr(Ⅵ) induces the dysfunction of mitochondrial respiratory chain in L-02hepatocytes
L-02hepatocytes were processed for mitochondria extraction after Cr(Ⅵ) exposure. The indices of mitochondrial respiration such as state3and state4respiration rates, respiratory control rate (RCR) were measured using Clark oxygen electrode by recording oxygen consummation (expressed as nmol/min/mg protein). L-02hepatocytes were treated with Cr(Ⅵ)(0,4,8,16,32μM) for24h. The mitochondria were used to detect the activities of mitochondrial respiratory chain complex (MRCC) Ⅰ-Ⅴ using the related kits. RNA was extracted and reversely transcripted into DNA. The mRNA expression levels of MRCC Ⅰ-Ⅴ were measured using real-time PCR. The protein expression levels of MRCC Ⅰ-Ⅴ were also detected using Western bloting. The mitochondrial membrane potential (Δψm), PTP open rate and membrane permeability, and the activities of LDH, ALT and AST were also measured in L-02hepatocytes after Cr(Ⅵ) exposure. The cellular content of adenine nucleotides (ATP, ADP, AMP) in Cr(Ⅵ)-treated hepatocytes were measured using high performance liquid chromatography (HPLC), and ATP/ADP ratio and energy charge (EC) were also calculated.
3. The effect of the dysfunction of mitochondrial respiratory chain on Cr(Ⅵ)-induced apoptosis in L-02hepatocytes
L-02hepatocytes were treated with PBS or16,32μM Cr(Ⅵ) for24 h, incubated with fluoresent probe CM-H2DCFDA and then were processed for the determination of ROS production. MRCC Ⅰ inhibitor Rotenone (ROT) which can induce ROS accumulation was used as positive control. The reduction site of Cr(Ⅵ) in mitochondria was determined by measuring the Cr(Ⅵ) reduction rate after the mitochondria were exposed to different substrates of MRCC. The antioxidant NAC was used to inhibit ROS since we inferred that ROS plays an important role in Cr(Ⅵ)-induced hepatocytes toxicity. The effect of NAC pre-treatment on cellular ATP content, p53mRNA and protein levels, the apoptotic pathway and HSP expressions in L-02hepatocytes after different doses of Cr(Ⅵ) exposure were measured. Cell cycle distribution of hepatocytes was also examined by flow cytometry. In order to explore the reasons for Cr(Ⅵ)-induced cell cycle distribution changes, by using real-time PCR and western blotting, we checked the expression levels of S phase checkpoint proteins (Tof1, Mrc1) and S phase related genes (CDK2, Cyclin E), as well as G2/M phase checkpoint proteins (BubR1, Mad2) and G2/M phase related proteins (Cyclin B, CDC25). We also used p53specific inhibitor PFT-a to block the function of p53in order to confirm if Cr(Ⅵ)-induced cell cycle arrest is depend on p53. Thus we measured the effect of PFT-a on the expression levels of p53, Mrc1, BubR1and cell cycle distribution in L-02hepatocytes exposed to different doses of Cr(Ⅵ). In addition to p53, we also examined the effect of Cr(Ⅵ) on NF-kB, PI3K/AKT pathway with or without ROS treatment.
4. The effect of the dysfunction of mitochondrial respiratory chain on Cr(Ⅵ)-induced premature senescence
L-02hepatocytes were treated with PBS or10nM Cr(Ⅵ) twice a week for24h for a total period of4weeks. SA-β-Gal activity was measured to indicate the occurrence of premature senescence. Cell cycle distribution was determined by flow cytometry. The identification of apoptosis and premature senescence was achieved by checking the percentages of senescent, apoptotic, necrotic and growing cells in L-02hepatocytes after Cr(Ⅵ) exposure. L-02hepatocytes after4weeks of treatment were analyzed for ROS production utilizing oxidant-sensitive fluorogenic probe CM-H2DCFDA. We also used the MitoSOX probe to measure specifically the mitochondrial superoxide level since MRCC blockade leads generally to superoxide production. To investigate the mechanism for the elevated ROS level, we determined the activities of MRCC Ⅰ-Ⅳ. We also measured the effect of Cr(Ⅵ) on p53mRNA levels and phosphorylated protein levels, as well as senescence pathway (p53-p21WAF1/CIP1, Rb-p16INK4a) and pro-survival genes, S phase related genes in hepatocytes with or without NAC pre-treatment. We further checked if Cr(Ⅵ)-induced premature senescence was completely depend on p53function. The transfection system containing shRNA to p53was used to knockout p53in hepatocytes. The non-target scramble (Scr)-transfected and p53shRNA-transfected hepatocytes were treated with PBS or10nM Cr(Ⅵ) twice a week for24h for4consecutive weeks. We then measured the effect of p53knocking-out on the occurrence of premature senescence, the percentage of senescent cells, the expression levels of pro-survival genes and S phase related genes in L-02hepatocytes after Cr(Ⅵ) exposure.
Results
1. Cr(VI) induces apoptosis in hepatocytes
Cr(VI) induced a concentration-dependent loss of cell viability in L-02hepatocytes. Considering the survival rate should be in a proper range (>70%) and the demand of statistic analysis, we chose four concentrations of Cr(Ⅵ)(4,8,16,32μM) for the following experiments. The apoptotic hepatocytes showed the characteristic DNA fragement after Cr(Ⅵ) exposure. TUNEL assay confirmed that DNA damage was increased in a dose-dependent manner. Both the early stage and late stage of apoptitic cells were increased with the increasing Cr(Ⅵ) exposure. The caspase8related Fas apoptitc pathway as well as caspase-9related non-Fas apoptotic pathway were all participated in Cr(Ⅵ)-induced apoptosis. Cr(Ⅵ) targeted HSP70and HSP90to regulated caspase-3activity. The measurement of GSH, MDA and SOD levels revealed that Cr(Ⅵ) significantly affected cellular antioxidant system and the ability of hepatocytes to generate ROS.
2. Cr(Ⅵ) induces the dysfunction of mitochondrial respiratory
chain in L-02hepatocytes
Cr(Ⅵ) inhibited state3respiration and RCR, but had no effect on state4respiration. Different doses of Cr(Ⅵ) exposure significantly inhibited mRNA and protein levels of MRCC Ⅰ and Ⅱ, especially the former. In the Cr(Ⅵ)-treated hepatocytes, Δψm was decreased, PTP open rate and the membrane permeability was increased. Cr(Ⅵ) also significantly reduced cellular ATP content, ATP/ADP ratio and EC.
3. Effect of mitochondrial respiratory chain dysfunction on apoptosis in Cr(Ⅵ)-treated hepatocytes
Cr(Ⅵ) induced ROS accumulation in L-02hepatocytes in a dose-dependent manner. Cr(Ⅵ) reduction rate significantly increased in the mitochondria treated with MRCC Ⅰ substrates glutamate/malate (Glu/Mal), indicating that Cr(Ⅵ) reduction occurs at MRCC I. NAC treatment significantly blocked the induction of apoptosis by inhibiting ROS accumulation, rescuing ATP reduction and inhibiting the activation of apoptic pathway. p53expression levels was increased in Cr(Ⅵ)-treated groups in a dose-dependent manner, while NAC treatment significantly blocked p53. Cr(Ⅵ) activated caspase-3by targeting ROS to regulate heat shock proteins. Low dose of Cr(Ⅵ)(4μM) exposure induced S phase cell cycle arrest while higher dose of Cr(Ⅵ)(16,32μM) exposure induced G2/M phase cell cycle arrest in L-02hepatocytes. Further investigation revealed that Cr(Ⅵ) could target S phase checkpoint proteins (Tof1、Mrc1) and S phase related proteins (CDK2, Cyclin E) to induce S phase arrest, and target G2/M phase checkpoint proteins (BubR1、Mad2) and G2/M phase related proteins (Cyclin B、CDC25) to induce G2/M phase arrest. NAC and PFT-a pre-treatment significantly alleviated Cr(Ⅵ)-induced S phase or G2/M phase cell cycle arrest, indicating that Cr(Ⅵ) could target ROS-p53to induce cell cycle arrest. In addition to p53, ROS also regulated NF-kB and PI3K/AKT pathway to induce apoptosis in L-02hepatocytes after Cr(Ⅵ) exposure.
4. Effect of mitochondrial respiratory chain dysfunction on premature senescence in Cr(Ⅵ)-treated hepatocytes
Low dose and long-term exposure of Cr(Ⅵ) induces premature senescence in L-02hepatocytes. Senescent cells showed significant S phase cell cycle arrest and the increase of SA-β-Gal activity. Similar to ROS production mechanisms in apoptotic cells, Cr(Ⅵ) could selectively target the sensitive sites MRCC Ⅰ and Ⅱ in respiratory chain to induce ROS accumulation. After Cr(Ⅵ) treatment, p53expression significantly increased in the senescent cells and phosphorylation of p53at Ser15also exhibited a clear increase. NAC pre-treatment resulted in almost undetectable p53expression in both the control and Cr(Ⅵ) treatment group, indicating the role of ROS played in regulating p53expression. Western blotting for senescence pathway analysis revealed that p53-p21WAF1/CIP1pathway, but not Rb-p16INK4a pathway was involved in Cr(Ⅵ)-induced premature senescence. In senescent cells, ROS could target p53to regulate the prosurvival genes Bcl-2, Mcl-2to inhibit cell proliferation, and also to regulate S phase related proteins CDK2, Cyclin E to induce S phase cell cycle arrest. The pre-treatment of NAC significantly inhibit the occurrence of premature senescence in hepatocytes after Cr(Ⅵ) exposure. Knocking-out of p53in L-02hepatocytes blocked the occurrence of premature senescence. Although premature senescence was not detected, ROS was found accumulation in L-02-p53shRNA cells, which indicating that Cr(Ⅵ)-induced premature senescence is depend on ROS function, while ROS function is completely depend on p53.
Conclusion
1. Cr(Ⅵ) induces apoptosis in L-02hepatocytes. The caspase-8related Fas apoptitc pathway as well as caspase-9related non-Fas apoptotic pathway are all participated in Cr(Ⅵ)-induced apoptosis.
2. Cr(Ⅵ) induces the inhibition of mitochondrial respiratory chain and the disturbance of energy metabolism in L-02hepatocytes.
3. The dysfunction of mitochondrial respiratory chain lead to ROS accumulation. Cr(Ⅵ) targets and inhibits MRCC Ⅰ and Ⅱ to increase ROS cellular level, and the reduction of Cr(Ⅵ) occurs at MRCC I. Low dose of Cr(Ⅵ) exposure induces S phase cell cycle arrest while high dose of Cr(Ⅵ) exposure induces G2/M phase eel cycle arrest. Cr(Ⅵ)-induced apoptosis in L-02hepatocytes is depent on ROS-p53, NF-kB and PI3K/AKT pathway.
4. Low dose and long-term Cr(Ⅵ) exposure induces premature senescence in L-02hepatocytes. p53-p21WAF1/CIP1pathway was involved in Cr(Ⅵ)-induced premature senescence. ROS targets and regulates pro-survival genes and S phase related proteins to induced premature senescence. Cr(Ⅵ)-induced premature senescence is depent on ROS-p53function.
铬及其化合物已成为严重威胁人类健康的不可忽视的环境污染物,广泛应用于工业生产过程如冶金、电镀等。铬的职业暴露可引发的健康危害包括接触性皮炎、鼻中隔穿孔以及支气管癌等。Cr(Ⅵ)可引发急慢性肝炎以及肝损伤。Cr(Ⅵ)最主要的细胞毒作用是诱导凋亡的发生,虽然已有大量的研究证实ROS在Cr(Ⅵ)诱导的细胞毒性中发挥极为重要的作用,但Cr(Ⅵ)具体的作用机制仍不十分清楚,比如ROS如何产生的,Cr(Ⅵ)如何诱导p53、NF-kB的激活等等。本研究除了阐明Cr(Ⅵ)诱导肝细胞凋亡的作用机制外,也对其诱导肝细胞发生早衰进行了探讨。细胞早衰是近年来的研究热点,是-种不同于凋亡的细胞毒性结局。环境中Cr(Ⅵ)的人群暴露多为低剂量长期的暴露,故研究早衰具有极其重要的实际意义。早衰被认为是抑制细胞恶性转化的一道有力屏障,而凋亡是细胞的程序性死亡,故本研究对Cr(Ⅵ)诱导早衰与凋亡的同时探讨将对阐明Cr(Ⅵ)的致癌机制提供一定的实验依据。
研究方法
1.Cr(Ⅵ)诱导肝细胞凋亡
首先用MTT法检测不同浓度的Cr(Ⅵ)(0-512μM)对L-02肝细胞存活率的影响,选取适宜的浓度(存活率>70%)进行后续试验。DNA-Ladder法检测Cr(Ⅵ)暴露后DNA的损伤；末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)法对完整的单个凋亡细胞核或凋亡小体进行原位染色,反应细胞凋亡；Annexin V-FITC/PI双染法借助流式细胞仪检测Cr(Ⅵ)暴露后的早期和晚期凋亡细胞；用western blotting法检测不同浓度的Cr(Ⅵ)处理后对PARP、 Caspase-8、Caspase-9、Caspase-3蛋白表达水平的影响,进而分析细胞凋亡通路；比色法检测Cr(Ⅵ)对Caspase-3活性的影响；通过对热休克蛋白HSP70与HSP90表达水平的分析研究HSP与caspase-3的关系；通过检测GSH、MDA以及SOD研究Cr(Ⅵ)对细胞内抗氧化系统的影响以及产生自由基的能力。
2.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱
Cr(Ⅵ)处理后提取肝细胞线粒体用Clark氧电极通过记录氧耗(nmol/min/mg protein)来检测线粒体呼吸功能,指标包括三态呼吸、四态呼吸以及呼吸控制率等；用不同浓度的Cr(Ⅵ)(0、4、8、16、32μM)处理L-02肝细胞24h。提取线粒体用试剂盒检测线粒体呼吸链复合体I-V的活性；提取RNA并经逆转录成DNA后用real-timePCR法检测复合体I-VmRNA基因的表达；本研究同时用western blotting法检测了Cr(Ⅵ)对线粒体呼吸链复合体I-V蛋白表达水平的影响；同时检测了Cr(Ⅵ)对线粒体膜电位(△Ψm)、PTP孔开放度、膜通透性以及对肝细胞中LDH,ALT,AST活性的影响；高效液相色谱法检测Cr(Ⅵ)对肝细胞内腺苷酸含量(ATP、ADP、AMP)、ATP/ADP比值、以及能荷(EC)的影响。
3.Cr(Ⅵ)诱导的肝细胞线粒体呼吸链功能紊乱在凋亡中的作用
用PBS以及16、32μM Cr(Ⅵ)处理L-02肝细胞24h,经荧光探针CM-H2DCFDA孵育后在荧光显微镜下检测ROS的生成情况,同时用已被证实可诱导ROS生成增加MRCC I抑制剂ROT用做阳性对照；用不同MRCC的底物作用于肝细胞线粒体后测定Cr(Ⅵ)还原速率检测Cr(Ⅵ)在线粒体中的还原位点；本研究推断ROS在Cr(Ⅵ)诱导的L-02肝细胞毒性中发挥了重要的作用,故用抗氧化剂NAC抑制ROS来证实；检测NAC对不同浓度Cr(Ⅵ)暴露后的肝细胞ATP含量的影响；检测Cr(Ⅵ)暴露后对p53mRNA以及蛋白表达水平的影响以及NAC的作用；检测NAC预处理对不同浓度的Cr(Ⅵ)处理后热休克蛋白表达的影响；流式细胞仪检测肝细胞细胞周期的分布情况；为阐明Cr(Ⅵ)诱导的L-02肝细胞细胞周期分布变化的原因,本研究用荧光定量PCR法以及western blotting法检测了Cr(Ⅵ)作用后细胞S期检查点蛋白(Tof1、Mrc1)以及S期相关基因(CDK2,Cyclin E)的表达水平,同时也检测了G2/M期检查点蛋白(BubR1、Mad2)以及相关基因(Cyclin B、CDC25)表达水平的影响；未验证Cr(Ⅵ)诱导的细胞周期阻滞是否与p53相关,本研究选用特异性的p53抑制剂PFT-a来阻断p53的作用；检测PFT-a对不同浓度Cr(Ⅵ)处理组L-02肝细胞中p53、Mrc1、BubR1以及细胞周期分布的影响；除p53外,本研究也检测了Cr(Ⅵ)对NF-κB、PI3K/AKT通路的影响以及ROS在其中的作用。
4.Cr(Ⅵ)诱导的肝细胞线粒体呼吸链功能紊乱在早衰中的作用
用PBS或10nM的Cr(Ⅵ)处理L-02肝细胞,每周2次,每次24h,持续4周；SA-B-Gal染色检测早衰的发生；流式细胞仪细胞周期分布的分析；检测Cr(Ⅵ)对早衰L-02肝细胞中衰老、坏死、凋亡以及增殖细胞比例的影响,从而鉴别早衰与凋亡；用氧化敏感型荧光探针CM-H2DCFDA在荧光显微镜下检测ROS的生成情况；由于线粒体呼吸链复合体的抑制通常引发线粒体超氧化物的产生,本研究用线粒体超氧化物探针MitoSOX Red特异性标记超氧化物的产生情况；检测Cr(Ⅵ)诱导的早衰L-02肝细胞中线粒体呼吸链复合体(MRCC) Ⅰ-Ⅴ活性；检测有或无NAC预处理作用时Cr(Ⅵ)对肝细胞中p53mRNA表达水平的影响；Cr(Ⅵ)诱导的早衰L-02肝细胞中p53磷酸化蛋白的检测；有或无NAC预处理作用时Cr(Ⅵ)对肝细胞中衰老通路(p53-p21WAF1/CIP1、Rb-p16INK4a)蛋白水平以及对肝细胞中促存活基因以及细胞周期S期相关蛋白表达水平的影响；为证实是否ROS诱导细胞发生早衰的功能完全依赖于p53的功能,本研究用脂质体转染在肝细胞中敲除p53,用无靶基因作用的Scramble(Scr)做为对照；用PBS或10nM的Cr(Ⅵ)处理L-02-Scr以及L-02-p53shRNA,每周2次,每次24h,持续4周；检测p53敲除对Cr(Ⅵ)诱导L-02肝细胞早衰发生以及检测衰老细胞百分比的影响；检测p53敲除对Cr(Ⅵ)暴露后肝细胞中促存活基因以及细胞周期S期相关蛋白表达水平的影响。
研究结果
1.Cr(Ⅵ)诱导肝细胞凋亡
Cr(Ⅵ)对L-02肝细胞存活率的抑制存在明显的剂量-效应依存关系,为保证细胞存活率在一个比较适宜的范围(>70%)以及考虑到统计学分析的需要,本研究各个指标检测选用Cr(Ⅵ)的处理浓度为4、8、16、32μM。Cr(Ⅵ)处理后的肝细胞呈现凋亡细胞特征性的梯状DNA条带。TUNEL实验证实Cr(Ⅵ)处理后损伤的DNA片段增加,即Cr(Ⅵ)可以诱导L-02肝细胞发生凋亡,且凋亡的细胞数目随着Cr(Ⅵ)染毒浓度的增加而增加。Cr(Ⅵ)处理后无论是凋亡细胞或是晚期凋亡坏死细胞都随Cr(Ⅵ)染毒浓度的增加而增加,呈现明显剂量-效应关系。Caspase-8参与的死亡受体依赖性凋亡通路以及Caspase-9参与的非死亡受体依赖性凋亡通路均参与到了Cr(Ⅵ)诱导的肝细胞凋亡之中。Cr(Ⅵ)通过抑制HSP70与HSP90的表达水平而影响caspase-3的活性。对GSH、MDA以及SOD的检测结果说明Cr(Ⅵ)明显影响细胞内抗氧化系统以及自由基产生能力。
2.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱
Cr(Ⅵ)抑制线粒体三态呼吸而并未对四态呼吸产生影响,呼吸控制率RCR明显降低。不同浓度的Cr(Ⅵ)处理显著抑制了MRCCⅠ和Ⅱ的活性、mRNA以及蛋白表达水平,尤其是MRCCⅠ。Cr(Ⅵ)处理后,线粒体的膜电位降低、PTP开放度增大、膜的通透性升高,酶类外溢。Cr(Ⅵ)降低肝细胞内腺苷酸含量、ATP/ADP比值以及EC。
3.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱在凋亡中的作用
Cr(Ⅵ)处理组肝细胞ROS生成量随着染毒浓度的增加而增加。用MRCCⅠ的底物谷氨酸以及苹果酸(Glu+Mal)处理肝细胞线粒体时Cr(Ⅵ)的还原速率均明显增大,说明Cr(Ⅵ)在MRCCⅠ处被还原。NAC阻断了各个Cr(Ⅵ)处理组中ROS的堆积,肝细胞中ATP降低趋势有明显地缓解,抑制细胞凋亡通路的激活从而抑制了肝细胞凋亡的发生。Cr(Ⅵ)处理组细胞p53蛋白表达水平明显增加,且呈现明显的剂量-效应关系,而NAC明显抑制了p53mRNA水平的表达。Cr(Ⅵ)是通过调节ROS进而调节热休克蛋白的表达而激活caspase-3。低浓度Cr(Ⅵ)(4μM)处理组细胞发生明显的S期细胞周期阻滞,而高浓度Cr(Ⅵ)(16、32gM)处理组细胞发生明显的G2/M期细胞周期阻滞。进一步研究发现低浓度Cr(Ⅵ)导致L-02肝细胞发生S期细胞阻滞的原因可能为诱导S期检查点蛋白基因(Tof1、Mrc1)以及S期相关基因(CDK2、Cyclin E)表达水平下降有关；而高浓度Cr(Ⅵ)导致L-02肝细胞发生G2/M期细胞阻滞的原因可能为诱导G2/M期检查点蛋白基因(BubR1、Mad2)以及S期相关基因(Cyclin B、CDC25)表达水平下降有关。NAC以及PFT-a预处理明显缓解了细胞的S期或G2/M期阻滞,说明Cr(Ⅵ)通过调控ROS水平进而调控p53而诱导L-02肝细胞细胞周期阻滞。Cr(Ⅵ)诱导的ROS堆积除作用于p53外也作用于NF-kB与PI3K/AKT通路,参与细胞毒作用。
4.Cr(Ⅵ)诱导肝细胞线粒体呼吸链功能紊乱在早衰中的作用
低剂量长期Cr(Ⅵ)暴露引发L-02肝细胞发生早衰。早衰细胞出现明显的S期细胞周期阻滞,SA-β-Gal活性明显增高。与Cr(Ⅵ)诱导肝细胞凋亡中ROS的生成机制类似,早衰细胞中Cr(Ⅵ)可以选择性作用于线粒体呼吸链的易感位点MRCCⅠ和MRCC Ⅱ而诱导ROS的大量堆积。衰老细胞中磷酸化p53(ser15)明显升高,p53被明显激活, NAC预处理明显降低了对照组和早衰细胞中p53水平,提示ROS可以对p53进行转录调控。对Cr(Ⅵ)诱导的早衰肝细胞中衰老通路的分析发现p53-p21WAF1/CIP1通路,而不是Rb-p16INK4a通路参与了肝细胞早衰的过程。早衰细胞中ROS调节p53后进而调节促存活基因Bcl-2、Mcl-1的表达而抑制肝细胞的增殖能力,通过调节S期相关蛋白CDK2、Cyclin E进而诱导S期细胞周期阻滞。NAC预处理明显抑制了细胞早衰的发生。肝细胞中敲除p53后发现p53的缺失抑制了细胞早衰的发生。虽然Cr(Ⅵ)处理后的L-02-p53shRNA细胞未发生早衰,但其ROS生成量仍明显增高,证实Cr(Ⅵ)诱导的L-02细胞发生早衰依赖于ROS,而ROS功能的发挥完全依赖于p53。
研究结论
1.Cr(Ⅵ)诱导L-02肝细胞发生凋亡,且Caspase-8介导的死亡受体依赖性凋亡通路以及Caspase-9介导的非死亡受体依赖性凋亡通路均参与其中。Caspase-3的活化与热休克蛋白(HSP70,HSP90)的抑制有关。
2.Cr(Ⅵ)诱导肝细胞线粒体呼吸链的抑制以及能量代谢障碍,表现为三态呼吸的抑制、呼吸控制率的降低和ATP的耗竭等。
3.线粒体呼吸链功能紊乱的直接结果是ROS的大量堆积。Cr(Ⅵ)通过抑制线粒体呼吸链复合体Ⅰ、Ⅱ的活性而诱导ROS水平剧增,且Cr(Ⅵ)在MRCC Ⅰ处被还原。低浓度或高浓度Cr(Ⅵ)处理分别发生S期或G2/M期细胞周期阻滞,其机制与Cr(Ⅵ)作用后ROS调控p53,后者进一步调控细胞周期检查点蛋白以及细胞周期相关蛋白有关。Cr(Ⅵ)诱导的细胞凋亡依赖于ROS-p53、NF-κB以及PI3K/AKT通路。4.低剂量长期Cr(Ⅵ)暴露引发L-02肝细胞发生早衰,以不可逆性的S期细胞周期阻滞以及SA-β-Gal活性的明显增高为特征。p53-p21WAF1/CIP1通路参与早衰过程。ROS通过调节促存活基因以及S期相关蛋白而诱导S期周期阻滞。敲除p53的肝细胞经Cr(Ⅵ)诱导后不发生早衰,提示早衰的发生完全依赖于ROS-p53功能。
Background
Chromium and its compounds are non-negligible pollutant that have became a serious threat to public health in the world with increasing use in diverse industrial processes, including metallurgy, electroplating, leather tanning and chroming. Occupational exposure to chromium is associated with several adverse effects of health, such as contact dermatitis, nasal perforation, and bronchiogenic cancer. Cr(Ⅵ) can induce toxic and chronic hepatitis and severe liver damage. The main cytotoxicity of Cr(Ⅵ) is apoptosis induction. Although it is believed that ROS plays an important role in the toxicity of Cr(Ⅵ), the related mechanisms still remain unclear, for example, how and where ROS are produced, how does Cr(Ⅵ) induce the activation of p53and NF-kB? In addition to explore the mechanisms for Cr(Ⅵ)-induced apoptosis, we also placed high emphasis on Cr(Ⅵ)-induce premature senescence. Premature senescence, which has been studied a lot recently, is totally different from apoptosis. The occupational exposure to Cr(Ⅵ) is low dose and long term, that is why the research on Cr(Ⅵ)-induced premature senescence has great significance. Premature senescence can be also viewed as an intrinsic cellular barrier against tumorigenesis, while apoptosis is programmed cell death. The present research which focused on both the apoptisis and premature senescence, has depicted an original cellular toxic phenomenon in response to Cr(Ⅵ) that may constitute a protective mechanism to the tumorigenic effect of chromium in humans.
Methods
1. Cr(Ⅵ) induces apoptosis in L-02hepatocytes
Using MTT method to detect the effect of different doses of Cr(Ⅵ)(0-512μM) exposure on L-02hepatocytes survival rate, and choose proper Cr(Ⅵ) treated concentrations (survival rate>70%) for the following experiments. Cr(Ⅵ)-induced DNA damage was examined by DNA-Ladder method. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used to detect the occurrence of apoptosis in the cells. Annexin V-FITC/PI double stain was used to detect the apoptotic cells in both the early stage and late stage. Western blotting was processed in the study to detect the effect of Cr(Ⅵ) on the expression levels of PARP, Caspase-8, Caspase-9and Caspase-3to further analyze the apoptic pathway. The activity of Caspase-3was also measured using colorimetry. By analyzing the expression levels of heat shock proteins HSP70and HSP90after Cr(Ⅵ) exposure to explore the correlation between HSP and caspase-3.By detecting the levels of GSH, MDA and SOD to study the effect of Cr(Ⅵ) on cellular antioxidant system and the capability of the hepatocytes to produce oxygen radicals.
2Cr(Ⅵ) induces the dysfunction of mitochondrial respiratory chain in L-02hepatocytes
L-02hepatocytes were processed for mitochondria extraction after Cr(Ⅵ) exposure. The indices of mitochondrial respiration such as state3and state4respiration rates, respiratory control rate (RCR) were measured using Clark oxygen electrode by recording oxygen consummation (expressed as nmol/min/mg protein). L-02hepatocytes were treated with Cr(Ⅵ)(0,4,8,16,32μM) for24h. The mitochondria were used to detect the activities of mitochondrial respiratory chain complex (MRCC) Ⅰ-Ⅴ using the related kits. RNA was extracted and reversely transcripted into DNA. The mRNA expression levels of MRCC Ⅰ-Ⅴ were measured using real-time PCR. The protein expression levels of MRCC Ⅰ-Ⅴ were also detected using Western bloting. The mitochondrial membrane potential (Δψm), PTP open rate and membrane permeability, and the activities of LDH, ALT and AST were also measured in L-02hepatocytes after Cr(Ⅵ) exposure. The cellular content of adenine nucleotides (ATP, ADP, AMP) in Cr(Ⅵ)-treated hepatocytes were measured using high performance liquid chromatography (HPLC), and ATP/ADP ratio and energy charge (EC) were also calculated.
3. The effect of the dysfunction of mitochondrial respiratory chain on Cr(Ⅵ)-induced apoptosis in L-02hepatocytes
L-02hepatocytes were treated with PBS or16,32μM Cr(Ⅵ) for24 h, incubated with fluoresent probe CM-H2DCFDA and then were processed for the determination of ROS production. MRCC Ⅰ inhibitor Rotenone (ROT) which can induce ROS accumulation was used as positive control. The reduction site of Cr(Ⅵ) in mitochondria was determined by measuring the Cr(Ⅵ) reduction rate after the mitochondria were exposed to different substrates of MRCC. The antioxidant NAC was used to inhibit ROS since we inferred that ROS plays an important role in Cr(Ⅵ)-induced hepatocytes toxicity. The effect of NAC pre-treatment on cellular ATP content, p53mRNA and protein levels, the apoptotic pathway and HSP expressions in L-02hepatocytes after different doses of Cr(Ⅵ) exposure were measured. Cell cycle distribution of hepatocytes was also examined by flow cytometry. In order to explore the reasons for Cr(Ⅵ)-induced cell cycle distribution changes, by using real-time PCR and western blotting, we checked the expression levels of S phase checkpoint proteins (Tof1, Mrc1) and S phase related genes (CDK2, Cyclin E), as well as G2/M phase checkpoint proteins (BubR1, Mad2) and G2/M phase related proteins (Cyclin B, CDC25). We also used p53specific inhibitor PFT-a to block the function of p53in order to confirm if Cr(Ⅵ)-induced cell cycle arrest is depend on p53. Thus we measured the effect of PFT-a on the expression levels of p53, Mrc1, BubR1and cell cycle distribution in L-02hepatocytes exposed to different doses of Cr(Ⅵ). In addition to p53, we also examined the effect of Cr(Ⅵ) on NF-kB, PI3K/AKT pathway with or without ROS treatment.
4. The effect of the dysfunction of mitochondrial respiratory chain on Cr(Ⅵ)-induced premature senescence
L-02hepatocytes were treated with PBS or10nM Cr(Ⅵ) twice a week for24h for a total period of4weeks. SA-β-Gal activity was measured to indicate the occurrence of premature senescence. Cell cycle distribution was determined by flow cytometry. The identification of apoptosis and premature senescence was achieved by checking the percentages of senescent, apoptotic, necrotic and growing cells in L-02hepatocytes after Cr(Ⅵ) exposure. L-02hepatocytes after4weeks of treatment were analyzed for ROS production utilizing oxidant-sensitive fluorogenic probe CM-H2DCFDA. We also used the MitoSOX probe to measure specifically the mitochondrial superoxide level since MRCC blockade leads generally to superoxide production. To investigate the mechanism for the elevated ROS level, we determined the activities of MRCC Ⅰ-Ⅳ. We also measured the effect of Cr(Ⅵ) on p53mRNA levels and phosphorylated protein levels, as well as senescence pathway (p53-p21WAF1/CIP1, Rb-p16INK4a) and pro-survival genes, S phase related genes in hepatocytes with or without NAC pre-treatment. We further checked if Cr(Ⅵ)-induced premature senescence was completely depend on p53function. The transfection system containing shRNA to p53was used to knockout p53in hepatocytes. The non-target scramble (Scr)-transfected and p53shRNA-transfected hepatocytes were treated with PBS or10nM Cr(Ⅵ) twice a week for24h for4consecutive weeks. We then measured the effect of p53knocking-out on the occurrence of premature senescence, the percentage of senescent cells, the expression levels of pro-survival genes and S phase related genes in L-02hepatocytes after Cr(Ⅵ) exposure.
Results
1. Cr(VI) induces apoptosis in hepatocytes
Cr(VI) induced a concentration-dependent loss of cell viability in L-02hepatocytes. Considering the survival rate should be in a proper range (>70%) and the demand of statistic analysis, we chose four concentrations of Cr(Ⅵ)(4,8,16,32μM) for the following experiments. The apoptotic hepatocytes showed the characteristic DNA fragement after Cr(Ⅵ) exposure. TUNEL assay confirmed that DNA damage was increased in a dose-dependent manner. Both the early stage and late stage of apoptitic cells were increased with the increasing Cr(Ⅵ) exposure. The caspase8related Fas apoptitc pathway as well as caspase-9related non-Fas apoptotic pathway were all participated in Cr(Ⅵ)-induced apoptosis. Cr(Ⅵ) targeted HSP70and HSP90to regulated caspase-3activity. The measurement of GSH, MDA and SOD levels revealed that Cr(Ⅵ) significantly affected cellular antioxidant system and the ability of hepatocytes to generate ROS.
2. Cr(Ⅵ) induces the dysfunction of mitochondrial respiratory
chain in L-02hepatocytes
Cr(Ⅵ) inhibited state3respiration and RCR, but had no effect on state4respiration. Different doses of Cr(Ⅵ) exposure significantly inhibited mRNA and protein levels of MRCC Ⅰ and Ⅱ, especially the former. In the Cr(Ⅵ)-treated hepatocytes, Δψm was decreased, PTP open rate and the membrane permeability was increased. Cr(Ⅵ) also significantly reduced cellular ATP content, ATP/ADP ratio and EC.
3. Effect of mitochondrial respiratory chain dysfunction on apoptosis in Cr(Ⅵ)-treated hepatocytes
Cr(Ⅵ) induced ROS accumulation in L-02hepatocytes in a dose-dependent manner. Cr(Ⅵ) reduction rate significantly increased in the mitochondria treated with MRCC Ⅰ substrates glutamate/malate (Glu/Mal), indicating that Cr(Ⅵ) reduction occurs at MRCC I. NAC treatment significantly blocked the induction of apoptosis by inhibiting ROS accumulation, rescuing ATP reduction and inhibiting the activation of apoptic pathway. p53expression levels was increased in Cr(Ⅵ)-treated groups in a dose-dependent manner, while NAC treatment significantly blocked p53. Cr(Ⅵ) activated caspase-3by targeting ROS to regulate heat shock proteins. Low dose of Cr(Ⅵ)(4μM) exposure induced S phase cell cycle arrest while higher dose of Cr(Ⅵ)(16,32μM) exposure induced G2/M phase cell cycle arrest in L-02hepatocytes. Further investigation revealed that Cr(Ⅵ) could target S phase checkpoint proteins (Tof1、Mrc1) and S phase related proteins (CDK2, Cyclin E) to induce S phase arrest, and target G2/M phase checkpoint proteins (BubR1、Mad2) and G2/M phase related proteins (Cyclin B、CDC25) to induce G2/M phase arrest. NAC and PFT-a pre-treatment significantly alleviated Cr(Ⅵ)-induced S phase or G2/M phase cell cycle arrest, indicating that Cr(Ⅵ) could target ROS-p53to induce cell cycle arrest. In addition to p53, ROS also regulated NF-kB and PI3K/AKT pathway to induce apoptosis in L-02hepatocytes after Cr(Ⅵ) exposure.
4. Effect of mitochondrial respiratory chain dysfunction on premature senescence in Cr(Ⅵ)-treated hepatocytes
Low dose and long-term exposure of Cr(Ⅵ) induces premature senescence in L-02hepatocytes. Senescent cells showed significant S phase cell cycle arrest and the increase of SA-β-Gal activity. Similar to ROS production mechanisms in apoptotic cells, Cr(Ⅵ) could selectively target the sensitive sites MRCC Ⅰ and Ⅱ in respiratory chain to induce ROS accumulation. After Cr(Ⅵ) treatment, p53expression significantly increased in the senescent cells and phosphorylation of p53at Ser15also exhibited a clear increase. NAC pre-treatment resulted in almost undetectable p53expression in both the control and Cr(Ⅵ) treatment group, indicating the role of ROS played in regulating p53expression. Western blotting for senescence pathway analysis revealed that p53-p21WAF1/CIP1pathway, but not Rb-p16INK4a pathway was involved in Cr(Ⅵ)-induced premature senescence. In senescent cells, ROS could target p53to regulate the prosurvival genes Bcl-2, Mcl-2to inhibit cell proliferation, and also to regulate S phase related proteins CDK2, Cyclin E to induce S phase cell cycle arrest. The pre-treatment of NAC significantly inhibit the occurrence of premature senescence in hepatocytes after Cr(Ⅵ) exposure. Knocking-out of p53in L-02hepatocytes blocked the occurrence of premature senescence. Although premature senescence was not detected, ROS was found accumulation in L-02-p53shRNA cells, which indicating that Cr(Ⅵ)-induced premature senescence is depend on ROS function, while ROS function is completely depend on p53.
Conclusion
1. Cr(Ⅵ) induces apoptosis in L-02hepatocytes. The caspase-8related Fas apoptitc pathway as well as caspase-9related non-Fas apoptotic pathway are all participated in Cr(Ⅵ)-induced apoptosis.
2. Cr(Ⅵ) induces the inhibition of mitochondrial respiratory chain and the disturbance of energy metabolism in L-02hepatocytes.
3. The dysfunction of mitochondrial respiratory chain lead to ROS accumulation. Cr(Ⅵ) targets and inhibits MRCC Ⅰ and Ⅱ to increase ROS cellular level, and the reduction of Cr(Ⅵ) occurs at MRCC I. Low dose of Cr(Ⅵ) exposure induces S phase cell cycle arrest while high dose of Cr(Ⅵ) exposure induces G2/M phase eel cycle arrest. Cr(Ⅵ)-induced apoptosis in L-02hepatocytes is depent on ROS-p53, NF-kB and PI3K/AKT pathway.
4. Low dose and long-term Cr(Ⅵ) exposure induces premature senescence in L-02hepatocytes. p53-p21WAF1/CIP1pathway was involved in Cr(Ⅵ)-induced premature senescence. ROS targets and regulates pro-survival genes and S phase related proteins to induced premature senescence. Cr(Ⅵ)-induced premature senescence is depent on ROS-p53function.
引文
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