Continuous in vitro exposure of intestinal epithelial cells to E171 food additive causes oxidative stress,inducing oxidation of DNA bases but no endoplasmic reticulum stress

The whitening and opacifying properties of titanium dioxide (TiO₂) are commonly exploited when it is used as a food additive (E171). However, the safety of this additive can be questioned as TiO₂ nanoparticles (TiO₂-NPs) have been classed at potentially toxic. This study aimed to shed some light on the mechanisms behind the potential toxicity of E171 on epithelial intestinal cells, using two in vitro models: (i) a monoculture of differentiated Caco-2 cells and (ii) a coculture of Caco-2 with HT29-MTX mucus-secreting cells. Cells were exposed to E171 and two different types of TiO₂-NPs, either acutely (6–48?h) or repeatedly (three times a week for 3 weeks). Our results confirm that E171 damaged these cells, and that the main mechanism of toxicity was oxidation effects. Responses of the two models to E171 were similar, with a moderate, but significant, accumulation of reactive oxygen species, and concomitant downregulation of the expression of the antioxidant enzymes catalase, superoxide dismutase and glutathione reductase. Oxidative damage to DNA was detected in exposed cells, proving that E171 effectively induces oxidative stress; however, no endoplasmic reticulum stress was detected. E171 effects were less intense after acute exposure compared to repeated exposure, which correlated with higher Ti accumulation. The effects were also more intense in cells exposed to E171 than in cells exposed to TiO₂-NPs. Taken together, these data show that E171 induces only moderate toxicity in epithelial intestinal cells, via oxidation.     

Hormetic Effects of Acute Methylmercury Exposure on Grp78 Expression in Rat Brain Cortex

This study aims to explore the expression of GRP78, a marker of endoplasmic reticulum (ER) stress, in the cortex of rat brains acutely exposed to methylmercury (MeHg). Thirty Sprague-Dawley (SD) rats were randomly divided into six groups, and decapitated 6 hours (h) after intraperitoneal (i.p.) injection of MeHg (2, 4, 6, 8 or 10 mg/kg body weight) or normal saline. Protein and mRNA expression of Grp78 were detected by western blotting and real-time PCR, respectively. The results showed that a gradual increase in GRP78 protein expression was observed in the cortex of rats acutely exposed to MeHg (2, 4 or 6 mg/kg). Protein levels peaked in the 6 mg/kg group (p < 0.05 vs. controls), decreased in the 8 mg/kg group, and bottomed below the control level in the 10 mg/kg group. Parallel changes were noted for Grp78 mRNA expression. It may be implied that acute exposure to MeHg induced hormetic dose-dependent changes in Grp78 mRNA and protein expression, suggesting that activation of ER stress is involved in MeHg-induced neurotoxicity. Low level MeHg exposure may induce GRP78 protein expression to stimulate endogenous cytoprotective mechanisms.     

非酒精性脂肪肝与钙稳态关系的研究进展

钙离子（Ca²⁺）是细胞内重要的第二信使,其主要储存在内质网和线粒体中,参与调控细胞多种生理功能及维持内质网、线粒体功能。内质网和线粒体对钙离子的摄取与释放直接影响胞内钙离子水平的变化,继而影响细胞正常生理功能。病理条件下,细胞内钙离子稳态失衡,可引发细胞生理功能异常并进一步影响内质网、线粒体功能。非酒精性脂肪肝（NAFLD）是临床常见病和多发病,其主要病理特征是：肝脏脂肪样变性、内质网应激、线粒体损伤和非特异性炎性反应等。其中持续的内质网应激及线粒体功能障碍是NAFLD发生发展的重要诱因。而细胞内钙稳态的变化可直接导致内质网及线粒体功能异常,继而影响NAFLD的发生发展。本文主要从钙稳态的主要影响因素以及钙稳态变化与NAFLD的关系两方面进行阐述,为寻求和研发防治NAFLD的药物提供新的方向。     

Endoplasmic reticulum may not be involved in the lead‐induced apoptosis in PC 12 cells in vitro

Recent researches indicated that mitochondrial pathway might play an important role in lead‐induced apoptosis. Our previous study also found that lead could induce apoptosis in PC 12 cells, and mitochondrial pathway events were involved in this process. As lead can disturb Ca²⁺ homeostasis, the present study was undertaken to determine whether lead can activate key cellular events in the endoplasmic reticulum (ER) pathway, including the expressions of C/EBP homology protein (CHOP) and glucose‐regulated protein 78 (GRP78), and the activation of caspase‐12 and calpain. The results showed that lead could increase the expression of GRP78, while the expressions of CHOP and procaspase‐12 remained unchanged. Moreover, the caspase‐12 and calpain were not activated, and the ultrastructure of endoplasmic reticulum was not altered. Therefore, it suggests that lead may induce apoptosis in PC 12 cells through mitochondrial pathway, but not through the endoplasmic reticulum pathway. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Effects of vinyltoluene alone and after pretreatment with polychlorinated biphenyls on hepatocellular morphology and enzyme activities in liver and kidneys in rats

Male Wistar rats were exposed by inhalation to vinyltoluene (300 ppm, 6 h daily, 5 days/week, for 1 or 2 weeks). An intraperitoneal dose of polychlorinated biphenyls (PCBs) (500 mg/kg) was injected 5 days before the inhalation exposure began. Hepatocytes were significantly increased in size after treatment with PCBs alone; this increase in size was caused by the proliferation of smooth endoplasmic reticulum (SER) and the dilatation of interreticular space. After treatment with vinyltoluene, the cell size decreased significantly. This decrease was counterbalanced by pretreatment with PCBs. Both with and without pretreatment with PCBs, the mitochondria were enlarged after exposure to vinyltoluene. Vinyltoluene affected the activity of NADPH-cytochrome c reductase and the content of cytochrome P-450 in the liver or the kidneys only moderately if at all. The activity of hepatic or renal O-deethylases (7-ethoxycoumarin and 7-ethoxyresorufin) and UDPglucuronosyltransferase increased 1,4-fold or more. The activity of epoxide hydrolase in the liver was increased 1.4-fold in 1 week by the inhalation of vinyltluene. A mixture of PCBs was a potent inducer of drug-metabolizing enzymes in the liver. A single i.p. injection of PCBs increased the activity of 7-ethoxyresorufin O-deethylase in the liver by more than 150 times. Exposure to both PCBs and vinyltoluene caused no additive effects on measured activities with the exception of hepatic epoxide hydrolase. The decrease in non-protein sulhydryl concentrations of the liver after exposure to vinyltoluene was accompanied by an increase in the excretion of urinary thioethers. In rats pretreated with PCBs, the effect of vinyltoluene on both of these parameters was less than that caused by the inhalaton of vinyltoluene alone. Our results indicate that the biotransformation of vinyltoluene in rats leads to glutathione conjugates accompanied by concomitant depletion of hepatic glutathione and increased excretion of urinary thioethers. Treatment with PCBs modifies the biotranformation of vinyltoluene in the direction of diol-formation via enhanced activity of epoxide hydrolase.     

内质网在铝致神经细胞凋亡过程中的作用

目的探讨内质网在铝致神经细胞凋亡过程中的作用。方法体外培养0~3 d新生大鼠的神经元细胞并用不同剂量氯化铝(AlCl3)染毒。(1)在光学显微镜和电子显微镜下观察神经元的凋亡和内质网的形态学改变;(2)测定凋亡相关蛋白Caspase和Ctyc在内质网的含量变化。结果(1)光学显微镜和电子显微镜形态学观察发现了凋亡的典型形态学改变,电子显微镜下可观察到内质网的变形肿胀及脱颗粒现象。(2)凋亡相关蛋白Caspase的含量随染毒剂量的加大而上升,二者具有相关性;在染毒后可检测到Ctyc由内质网释放到胞浆的过程。结论铝能诱导神经元凋亡,内质网在凋亡过程中发挥了重要的调控作用。     

内质网应激、自噬与凋亡在斑蝥素致大鼠肝毒性中的作用

目的 探讨斑蝥素(cantharidin，CTD)致大鼠药物性肝损伤(drug-induced liver injury，DILI)的毒理学机制。方法 采用不同剂量CTD(0.061 4，0.092 1，0.184 1 mg·kg⁻¹)连续灌胃SD大鼠 28 d，检测肝脏指数和血清肝功能指标，HE 染色评估肝脏病理变化。进一步采用免疫印迹法检测内质网应激(endoplasmic reticulum stress，ERS)、自噬和细胞凋亡通路蛋白。结果 CTD 干预后肝脏指数显著升高，生化指标ALT、AST、LDH、ALP和T-Bil显著升高， 且呈剂量依赖性，肝脏组织出现结构破坏和中央静脉扩张等病理变化；GRP78、CHOP、ATF4、Beclin-1、LC3、Caspase-3、Caspase-8和Bax/Bcl-2的蛋白表达水平显著升高。分子对接结果显示，GRP78、ATF4和Beclin-1与CTD对接结果良好。结论 CTD可激活大鼠ERS，进一步激活自噬，诱导下游凋亡，研究结果可为CTD诱导的DILI提供新的科学依据。     

昆布多糖对肾纤维化大鼠肾组织内质网应激的保护作用

目的研究昆布多糖对肾纤维化大鼠肾组织内质网应激(ERS)分子伴侣GRP78、GRP94蛋白表达的影响。方法采用单侧输尿管梗阻(UUO)诱导大鼠肾间质纤维化的动物模型,将大鼠随机分为假手术组、模型组、依那普利组、昆布多糖高、中、低剂量组。术后第7天处死大鼠,收集血清测定肌酐(Scr)、尿素氮(BUN)水平。采用Western免疫印迹法检测大鼠肾组织GRP78、GRP94的蛋白表达;采用HE、Masson染色检测肾小管损伤及肾间质纤维化程度。结果各治疗组与模型组比较大鼠肾小管间质损伤指数、肾间质纤维化程度、血清Scr、BUN水平及肾组织GRP78、GRP94蛋白表达有差异(P<0.05;P<0.01),昆布多糖与依那普利组比较,大鼠肾组织GRP78、GRP94表达升高(P<0.05),肾小管间质损伤及肾间质纤维化程度明显降低(P<0.05),肾功能Scr、BUN明显降低(P<0.05)。结论UUO早期昆布多糖可能通过上调ERS分子伴侣GRP78、GRP94蛋白表达,协助变性蛋白进行重新折叠、装配及跨膜转运,抑制未折叠蛋白反应,阻断ERS应激信号传导通路,从而减轻肾间质纤维化的发生和发展。     

辛伐他汀通过内质网应激途径诱导K562细胞凋亡

探讨内质网应激在辛伐他汀诱导K562细胞凋亡中的作用。采用荧光显微镜观察凋亡细胞的形态变化，AnnexinV-FITC/PI双染法检测细胞凋亡率，激光扫描共聚焦显微镜检测细胞内Ｃa²⁺浓度，RT-PCR检测葡萄糖调节蛋白78(glucose regulated protein 78，GRP78)、钙蛋白酶(calpain)基因mRNA表达水平，Western blotting检测GRP78、 calpain、 caspase-3， -6， -7， -9， -12蛋白水平。结果显示，10、 20、 30 μmol·L^-1辛伐他汀(simvastatin，Sim)作用K562细胞72 h后，细胞出现典型的凋亡形态，凋亡率分别为12.41%、 19.08%和23.41%；细胞内Ca²⁺浓度增加，荧光强度分别为43、54和64；GRP78、calpain基因mRNA表达上调；calpain、 caspase-3， -6， -7， -9， -12蛋白剪切活化、GRP78蛋白表达增强。以上结果表明，内质网作为细胞凋亡的重要途径参与了辛伐他汀诱导K562细胞的凋亡。辛伐他汀将可能被用于临床治疗白血病。     

ENDOPLASMIC RETICULUM STRESS INVOLVED IN HEART AND LIVER INJURY IN IRON-LOADED RATS

• 1 Iron overload contributes to the pathogenesis of various diseases and directly induces tissue injury. In the present study, we investigated the relationship between heart and liver injury induced by iron overload and cellular endoplasmic reticulum (ER) stress to explore the molecular mechanism of iron overload‐induced cellular injury.
• 2 Iron overload in rats was generated by intraperitoneal injection of iron–dextran chronically (30 mg/kg per day for 9 weeks) or acutely (300 mg/kg once). Tissue injury was assessed by determining serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, as well as malondialdehyde (MDA) content in the heart and liver. The ER stress response was analysed by expression of glucose‐response protein 78 (GRP78) and activation of caspase 12.
• 3 In chronic iron‐loaded rats, iron levels in the heart and liver were higher, by approximately 2‐and 7.8‐fold, respectively (P < 0.01), compared with control. Serum LDH, ALT and AST activity, as well as MDA content, GRP78 expression and caspase 12 activity in the heart and liver, were upregulated in chronically iron‐loaded rats. In acute iron‐loaded rats, iron content in the heart and liver was 51% and 63% higher than in controls (both P < 0.01). Serum LDH, ALT and AST activity, MDA content in the heart and liver and levels of ER stress markers were all increased in acute iron‐loaded rats. N‐Acetylcysteine (150 mg/kg, s.c.) lowered the levels of these parameters in acute iron‐loaded rats.
• 4 The results of the present study indicate that ER stress may play an important role in iron‐induced tissue injury and that reactive oxygen species may mediate the ER stress response in the pathogenesis of iron‐overload cellular injury.

     

愤怒诱导大鼠肝损伤中内质网应激相关蛋白的表达

目的:研究慢性愤怒诱导的大鼠肝损伤中内质网应激(ERS)相关蛋白的表达.方法:选择20只雄性SD大鼠,随机分为两组,各10只.实验组大鼠予应激刺激造模,对照组正常饲养,2周后同时处死两组大鼠.检测肝脏形态学变化及肝组织中GRP78、Caspas-3、LC3-Ⅱ、Beclin-1的mRNA和蛋白表达水平.结果:与对照组相...     

Effects of cyclopiazonic acid on the ultrastructure of rat liver

Groups of Sprague-Dawley rats were dosed per os for 4 consecutive days with 0.0, 0.2, 2.0 or 4.0 mg cyclopiazonic acid (CPA)/kg body weight/day, and killed on the fifth day. Sections of liver were prepared for electron microscopic examination. Dilatation of the rough endoplasmic reticulum was observed in all hepatocytes examined from the 2 highest dose groups, and in about 25% of liver cells from the 0.2 mg CPA/kg/day group. Vesiculation of the rough endoplasmic reticulum also occurred in these groups, an increasing amount of vesiculation being observed with increasing dosage. Control sections exhibited neither of these characteristics. No proliferation of smooth endoplasmic reticulum, or blockage of bile canaliculi was observed in any group. Lysing cells were present only in the 4.0 mg CPA/kg/day group; mitochondria in the 2.0 and 4.0 mg CPA/kg/day dose groups were swollen. Nuclei were ultrastructurally normal in all groups. The primary cellular effect of CPA was on the endoplasmic reticulum, even at relatively low doses. Possible interactions of CPA with other toxins likely to be produced by the same fungus, such as aflatoxin, are considered.     

Zearalenone Induces Apoptosis in Porcine Endometrial Stromal Cells through JNK Signaling Pathway Based on Endoplasmic Reticulum Stress

Zearalenone (ZEA) is an estrogen-like mycotoxin characterized mainly by reproductive toxicity, to which pigs are particularly sensitive. The aim of this study was to investigate the molecular mechanism of ZEA-induced apoptosis in porcine endometrial stromal cells (ESCs) by activating the JNK signaling pathway through endoplasmic reticulum stress (ERS). In this study, ESCs were exposed to ZEA, with the ERS inhibitor sodium 4-Phenylbutyrate (4-PBA) as a reference. The results showed that ZEA could damage cell structures, induce endoplasmic reticulum swelling and fragmentation, and decreased the ratio of live cells to dead cells significantly. In addition, ZEA could increase reactive oxygen species and Ca²⁺ levels; upregulate the expression of GRP78, CHOP, PERK, ASK1 and JNK; activate JNK phosphorylation and its high expression in the nucleus; upregulate the expression Caspase 3 and Caspase 9; and increase the Bax/Bcl-2 ratio, resulting in increased apoptosis. After 3 h of 4-PBA-pretreatment, ZEA was added for mixed culture, which showed that the inhibition of ERS could reduce the cytotoxicity of ZEA toward ESCs. Compared with the ZEA group, ERS inhibition increased cell viability; downregulated the expression of GRP78, CHOP, PERK, ASK1 and JNK; and decreased the nuclear level of p-JNK. The Bax/Bcl-2 ratio and the expression of Caspase 3 and Caspase 9 were downregulated, significantly alleviating apoptosis. These results demonstrate that ZEA can alter the morphology of ESCs, destroy their ultrastructure, and activate the JNK signaling via the ERS pathway, leading to apoptosis.     

Cisplatin, gentamicin, and p-aminophenol induce markers of endoplasmic reticulum stress in the rat kidneys.

In vitro evidence of the involvement of the endoplasmic reticulum (ER) during drug-induced renal toxicity is accumulating. ER stress and ER-mediated cell death markers have been reported after exposure of renal cells to model toxicants and nephrotoxic drugs in various in vitro models, but in vivo experiments with clinically relevant nephrotoxic compounds are lacking. In order to determine the relevance of the in vitro findings, markers of ER stress (XBP1 messenger RNA processing and protein expression; GRP78 and GRP94 upregulation) and ER-mediated cell death (caspase-12 and calpain activation) were examined in kidney tissue of rats exposed to nephrotoxic doses of cisplatin (CIS), gentamicin (GEN), and p-aminophenol (PAP), a nephrotoxic metabolite of acetaminophen. XBP1 signaling was observed with all three drugs and was associated with increased expression of GRP94 and GRP78 in GEN- and PAP-treated animals, but surprisingly not after CIS exposure. m-Calpain expression was increased after 7 days of CIS treatment, whereas it was decreased in PAP-treated rats. Caspase-12 cleavage products were increased after CIS, GEN, and PAP administration. The results of this study demonstrate that three clinically relevant nephrotoxic drugs are all associated with changes in markers of ER stress and ER-mediated cell death in vivo. Further investigation is warranted to determine the role of the ER, the calpain system, and caspase-12 in drug-induced renal cell death.     

Role of estrogen receptors in thyroid toxicity induced by mono (2-ethylhexyl) phthalate via endoplasmic reticulum stress: An in vitro mechanistic investigation

Mono(2-ethylhexyl) phthalate (MEHP) can influence the expression of estrogen receptors (ERs) and induce thyroid injury. The expression of ERs can be related to thyroid disease and abnormal expression of ERs has been associated with activation of endoplasmic reticulum stress. This study aimed to clarify the role of ERs in MEHP-induced thyroid damage via endoplasmic reticulum stress. We exposed Nthy-ori 3–1 cells to different doses of MEHP. We found that after the exposure, the cell viability and the expression levels of thyroid hormone metabolism-related proteins decreased, while the apoptosis level and the expression levels of ERs (ERα and GPR30) increased. Three endoplasmic reticulum stress-related signaling pathways were activated by MEHP. After ERα and GPR30 were knocked down, these three pathways were inhibited and the thyroid toxicity was alleviated. Taken together, our results indicate that MEHP can induce thyroid toxicity by upregulating the expression of ERs, further activating endoplasmic reticulum stress.     

内质网应激对小鼠卵巢卵泡发育的影响

摘要：目的 探索小鼠卵巢内质网应激对卵泡发育的影响。方法 对照组小鼠4只,实验组小鼠6只,应用经典 内质网应激诱导物衣霉素经腹腔注射诱导小鼠卵巢内质网应激激活,实时荧光定量逆转录聚合酶链反应（RT qPCR）检测未折叠蛋白质应答（UPR）标志分子HSPA5、CHOP、ATF4 mRNA表达情况,明确小鼠卵巢内质网应激激活 状态,苏木精伊红染色（HE染色）检测小鼠卵巢卵泡发育状态,明确内质网应激对小鼠卵巢卵泡发育的影响。结果 衣霉素的处理明显上调了小鼠卵巢内质网应激标志分子的表达,与对照组相比,衣霉素处理组小鼠卵巢卵泡发育明 显受限。结论 内质网应激的激活明显抑制了小鼠卵巢卵泡的发育     

内质网应激与心房颤动研究进展

心房颤动（AF）是临床常见的持续性心律失常,是卒中和心力衰竭的独立危险因素,然而其具体发病机制尚不完全清楚。内质网是调控蛋白质合成、细胞内Ca2+浓度、氧化应激水平、诱导细胞凋亡信号通路的主要细胞器,在心律失常发生和发展中的作用日益受到重视。多种致病因素可导致内质网应激（ERS）,其主要通过未折叠蛋白反应（UPR）来恢复内质网稳态。ERS的过度激活可导致心房肌细胞Ca2+超载、氧化应激失衡和细胞凋亡,在AF的发病机制中发挥重要作用。本文对ERS和AF研究进展进行了综述。     

17-甲氧基-7-羟基-苯并呋喃查尔酮对大鼠心肌缺血再灌注损伤的保护作用

目的观察17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)对心肌缺血再灌注(I/R)大鼠的影响。方法 SD大鼠50只随机分为5组,每组10只:假手术组,模型组,溶媒组,YLSC低、高剂量(2.50,5.00 mg/kg)组。缺血30 min再灌注60 min复制大鼠I/R模型,观察大鼠血清肌酸激酶(CK)、乳酸脱氢酶(LDH)、天门冬氨酸氨基转移酶(AST)含量的变化,伊文思蓝和氯化三苯四氮唑(TTC)双重染色确定心肌梗死面积,TUNEL法检测心肌细胞凋亡率,Western blot法检测心肌蛋白葡萄糖调节蛋白78(GRP78)和caspase12的表达。结果与模型组相比,YLSC能显著减少心肌梗死面积和CK、LDH、AST的漏出,降低心肌细胞凋亡率,并抑制内质网应激标志蛋白GRP78和caspase12的表达。结论 YLSC能减少大鼠I/R损伤,其心肌保护作用可能与减轻内质网应激有关。     

问荆硅化物对实验性肝损伤的保护作用

问荆硅化物(SCE)能降低正常大鼠的血清谷丙转氨酶(ALT)及CCl_4中毒大鼠升高的血清ALT,对CCl_4中毒小鼠升高的血清磺溴酞钠(BSP)滞留量也有明显降低作用;能显著降低硫代乙酰胺(TAA)及泼尼龙所致小鼠升高的血清ALT;能明显缩短戊巴此妥钠所致小鼠的睡眠时间,SCE还可使CCl_4中毒大鼠肝线粒体肿胀减轻,粗面内质网基本恢复正常,肝糖原颗粒增多,脂滴明显减少。