甲基汞对未分化型PC12细胞的毒性机制

应用未分化型PC12细胞为体外模型,研究甲基汞对多巴胺能神经元的毒性作用机制.利用MTT法检测甲基汞的细胞毒性,Hoechst33258荧光染色观察细胞形态变化,流式细胞术定量分析细胞周期分布和凋亡比率,并进一步通过比色法评价各项氧化应激指标.结果显示,甲基汞以剂量依赖性方式降低了细胞存活率,干扰细胞周期,造成S期细胞阻滞,细胞出现典型的凋亡性死亡特征.另外,甲基汞暴露导致PC12细胞内O-.2、H2O2和·OH等活性氧含量急剧增加,脂质过氧化程度升高,且SOD、CAT和GSH-Px等抗氧化酶活性降低.因此,甲基汞对多巴胺能神经元具有强烈的毒性,毒性作用机制涉及到干扰细胞周期、诱导细胞凋亡和引发严重的氧化损伤.     

Tetrachloro-1,4-benzoquinone induces apoptosis of mouse embryonic stem cells

Pentachlorophenol(PCP) is a widespread,persistent environmental contaminant,and it is enzymatically activated to form a reactive metabolite,tetrachloro-l,4-benzoquinone(TCBQ).To our knowledge,there is no information about TCBQ toxicity on embryonic stem cells.Here,we demonstrated that TCBQ induced significantly apoptosis of mouse embryonic stem cells in a concentration-dependent manner.We also showed that TCBQ elevated genomic5-hydroxymethylcytosine(5hmC) by affecting ten-eleven translocation(Tet) dioxygenases in mouse embryonic stem cells.We further investigated whether Tet dioxygenases were implicated in TCBQ-induced apoptosis.By depleting all three dioxygenases(Tet1-3),we found that Tet dioxygenases slightly inhibited both early and late apoptosis induced by TCBQ at a low concentration(30 μmol/L).Meanwhile,treated by TCBQ at higher concentrations(40and 50 μmol/L),the total percentage of apoptotic cells was not affected by Tet dioxygenases.However,Tet dioxygenases tended to arrest mouse ES cells to be at early apoptotic stage and to reduce the cells to enter later apoptotic stage.These results indicate that Tet dioxygenases play a role in shaping TCBQ-induced apoptosis in mouse embryonic stem cells.Our study provides new insights into the toxicology of PCP and its reactive metabolite TCBQ.     

苯胺对小鼠成纤维细胞L929的体外毒性评价

探讨不同浓度的苯胺化合物对小鼠成纤维细胞L929的体外毒性作用,为满足危险化学品分类鉴定和风险评估需求、更好地预防环境化合物对人体健康的损害提供理论依据。体外培养L929细胞,分别将不同浓度的苯胺化合物(0、0.001、0.01、0.1、1、10μg·mL~(-1))加入培养基中处理细胞,在不同时间点分别取材,MTT检测细胞增殖率,AO/PI染色检测细胞存活率,Annexin V-PI染色后流式细胞仪检测细胞凋亡,相关检测试剂盒测定细胞内活性氧(ROS)的含量及总谷胱甘肽的含量。结果表明,高浓度(1、10μg·mL~(-1))的苯胺化合物对L929细胞的生长和增殖具有显著的抑制效应。即使浓度降低至0.1μg·mL~(-1),在处理48 h后,细胞的存活数量显著下降,说明其对细胞增殖具有浓度和时间的累积效应和依赖性。同时,对各组细胞采用流式细胞仪测定细胞凋亡,发现低浓度的苯胺对细胞凋亡影响并不明显,但高浓度的苯胺处理后,细胞凋亡和坏死数量显著增加。ROS含量和GSH含量检测结果显示,较低浓度的苯胺对细胞氧化应激反应并无显著影响,但浓度至0.1μg·mL~(-1)时,细胞较对照组出现明显的氧化应激反应,说明细胞已受损,再继续培养将会导致细胞死亡。上述研究结果提示,高浓度苯胺化合物可显著影响L929细胞存活率,且具有时间和浓度累积效应。其毒性机制可能与细胞内活性氧增加、谷胱甘肽耗竭有关。     

杀虫单对不同倍性泥鳅鳍细胞系的毒性作用

以泥鳅鳍二倍体(DIMF)和三倍体(TRMF)细胞系为受试细胞,研究杀虫单对2种细胞系的毒性作用。采用噻唑蓝(MTT)法测得DIMF与TRMF 24 h半致死浓度分别为119.73 mg·L-1、146.26 mg·L~(-1)。DIMF的敏感性明显高于TRMF。经杀虫单处理的活体细胞表现为细胞伸长,空泡化和脱落并游离于培养基表面的现象。2种细胞系酶活力测定的结果显示:杀虫单浓度为0~100 mg·L~(-1)时,SOD和GST活力随着浓度的增加而增加,100~200 mg·L~(-1)浓度组酶活力逐渐降低;0~200 mg·L~(-1)时,ACh E活力与杀虫单浓度呈负相关,并且三倍体3种酶活力均高于二倍体。微核试验结果显示:50 mg·L~(-1)杀虫单就能对细胞核造成损伤并形成微核,微核率随杀虫单浓度增加而增加。当杀虫单浓度达到200 mg·L-1时,微核率出现最大值,DIMF和TRMF分别为3.3‰和3.7‰,2种细胞的测试结果无显著性差异(P0.05)。电镜观察结果显示:对照组DIMF和TRMF超微结构无明显差异;DIMF和TRMF病理变化情况相似:染色质凝集趋边,细胞核分解成多个,细胞内出现空泡和凋亡小体,表现出凋亡的特征。研究表明杀虫单的细胞毒性机制是通过改变细胞内酶活性从而诱导凋亡,不同倍性细胞系之间的差异主要与多倍体细胞体积大,胞内物质多,分裂快,生长旺盛等有关。     

The microcystins-induced DNA damage in the liver and the heart of zebrafish,Danio rerio

Microcystins (MCYST) are the freshwater cyanobacterial toxins, known to induce hepatocellular carcinoma, necrosis, intrahepatic bleeding, as well as human and livestock mortality. Within hepatocytes, MCYST selectively bind to protein phosphatases 1 and 2A, resulting in severe liver damage. The toxicology of MCYST in mice and rats has been well studied, but little is known regarding genotoxicity in aquatic animals. In this study, the zebrafish, Danio rerio was exposed to crude extract of Microcystis aeruginosa bloom. Liver and heart were examined for MCYST-induced toxicity. Light microscopy at 36?h revealed severe, widespread apoptotic necrosis of the majority of hepatocytes, and cytoskeletal deformation in myocardiocytes. Hepatocytes were dissociated with cell shrinkage and margination of nuclear chromatin. Laddering of genomic DNA from the liver and heart of the exposed fish in an increment of 180–200?bp was consistent with apoptosis. Fluorimetric analysis of DNA unwinding was carried out to determine the DNA strand breakage. After 36?h exposure, the % double-stranded DNA was significantly reduced in hepatocytes and myocardiocytes. In conclusion, the results obtained in this study indicate that, the extract of M. aeruginosa bloom is genotoxic to fish. The DNA damage observed in this study may be attributed to the activation of DNA endonucleases. This model of DNA damage may contribute for identifying novel molecular mechanisms of interest for therapeutic application.     

Cytotoxic and apoptotic activity of Scrophularia oxysepala in MCF-7 human breast cancer cells

Breast cancer is the most common cancer in women. Therefore, there is an urgent need to identify and develop therapeutic strategies against this deadly disease. This study is the first to investigate the cytotoxic and apoptotic effects of Scrophularia oxysepala in MCF-7 human breast cancer cells. Dichloromethane, methanol and n-hexane extracts of Scrophularia oxysepala were examined. The 3-(4,5-dimethylethiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assays were performed in MCF-7 cells as well as control cell line MCF10A to analyze the cytotoxic activity of the plant. Furthermore, the apoptosis-inducing action of extracts of the plant was evaluated using cell death detection ELISA, the terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay, Western blotting and real-time polymerase chain reaction (PCR). The results showed that dichloromethane and methanol extracts significantly inhibited cell growth and viability without inducing damage to MCF10A cells. Cell death detection ELISA, morphological changes of cells in TUNEL assay, Western blot detection of poly (ADP-ribose) polymerase (PARP) cleavage and increase in the mRNA expression levels of p-53, caspase-3 and c-myc showed that the induction of apoptosis was the main mechanism of cell death. Our results strongly suggest that this plant may contain potential bioactive compound(s) for the treatment of breast cancer.     

Low concentration of HgCl2 drives rat hepatocytes to autophagy/apoptosis/necroptosis in a time-dependent manner

Mercury (Hg) is known to produce hepatotoxicity driving cells towards apoptosis. It was recently reported that low concentrations of Hg (5 μM) initiate autophagy in vitro within 30 min of incubation modulated by several autophagy-related gene proteins, and co-regulators through ubiquitination. The present study aimed to elucidate in vitro mode of cytotoxic responses including programed cell death in 5-μM Hg-treated rat hepatocytes. Autophagy proceeded from 30 min to 4 hr mediated by crosstalk between specific regulating factors of cell-death-signaling mechanisms. It was noted that after 4-hr incubation with 5 μM HgCl₂, cells were driven towards apoptosis followed by necroptosis within 6.5 hr. Receptor-interacting serine-threonine protein 3 (RIP3) and caspase-8 played a significant role in interlinking function. The positive role of caspase-8 with RIP3 significantly triggered caspase-3 via extrinsic apoptotic pathway. A shift from apoptosis to necroptosis occurred after 6 hr via tumor necrosis factor α-RIP3-caspase-8 pathway. No alteration in caspase-3 expression and presence of high-mobility group box 1 protein in nucleus indicated absence of apoptosis and necrosis in rat hepatocytes between 6.5 and 8 hr. Data indicated that cellular homeostasis is regulated by modulating different proteins and driving hepatocytes through autophagy to apoptosis to necroptosis in a time-dependent manner.     

Dietary bioflavonoid quercetin modulates porcine ovarian granulosa cell functions in vitro

Quercetin is a dietary bioflavonoid used widely as a food supplement and is generally recognized as safe. The aim of this in vitro study was to examine the steroid hormone (progesterone and 17- β estradiol) release, proliferation (PCNA and cyclin B1) and apoptosis (caspase 3 and p53) of porcine ovarian granulosa cells after the addition of quercetin at concentrations 0.01, 0.1, 1, 10 and 100?μmol?L^?1. Progesterone release was stimulated at the concentration 10?μmol?L^?1. Quercetin neither had any impact on 17-β estradiol secretion nor on the presence of PCNA. However, a significant enhancement of the occurrence of cyclin B1 was noted except for the lowest concentration 0.01?μmol?L^?1. Quercetin did not have any influence on the number of granulosa cells containing caspase 3, but at the concentration 10?μmol?L^?1 it inhibited p53 occurrence. Results confirm the safety of quercetin in porcine ovarian granulosa cell model and further suggest its possible concentration-dependent influence on ovarian functions through pathway that may involve progesterone, cyclin B1 and p53.     

磁铁矿纳米颗粒与Pb~(2+)复合物对大鼠肾细胞的毒性研究

磁铁矿纳米颗粒(magnetite nanoparticles, MNPs)是一种环境友好型吸附剂,广泛应用于废水中Pb~(2+)的处理。目前,有不少关于MNPs毒性的研究,但对MNPs处理Pb~(2+)形成的复合物的毒性却鲜有报道,其复合毒性亟待深入研究。本文以大鼠肾细胞(NRK)作为细胞模型,系统研究不同Pb含量的MNPs-Pb复合物,以及相应浓度的MNPs和Pb~(2+),对大鼠肾细胞活性、细胞形态和密度的影响,考察细胞对纳米颗粒的摄取以及细胞凋亡的作用机制,评估MNPs-Pb的毒性效应。结果表明,在本实验浓度和暴露时间(12 h)条件下,Pb~(2+)能够显著抑制细胞活性,改变细胞形态,促进细胞凋亡,对细胞有显著的毒性效应,并且呈现剂量相关性;利用MNPs吸附水环境中Pb~(2+)形成的复合物MNPs-Pb对大鼠肾细胞没有显著的毒性作用(P<0.05),MNPs对Pb~(2+)的吸附可能是Pb~(2+)的细胞毒性降低的原因。     

Carbon black-benzo(a)pyrene complex-induced apoptosis and autophagy blockage in rat alveolar macrophages

Abstract

Cooking of foods and the burning of biomass and fossil fuels in stoves are the main sources of cooking fumes, with carbon black and polycyclic aromatic hydrocarbons as main components. The toxicity of carbon black and polycyclic aromatic hydrocarbons has been well studied individually, but the combined toxicity is much less understood. Carbon black can adsorb benzo(a)pyrene to form a complex which displays an altered physical form which in this study has been constructed to simulate particles in the cooking fumes and explore the combined toxic effect on rat alveolar macrophages. The complex-induced cell apoptosis and blocked cell autophagy flux compared with both individually. The mechanism of toxicity may be by intracellular reactive oxygen species generation, impairing the mitochondrial membrane potential and activating apoptosis signaling pathways.