抗氧化剂对砷诱导人膀胱上皮细胞氧化应激相关通路的影响

目的研究抗氧化剂对亚砷酸钠诱导的人膀胱上皮细胞系(SV-HUC-1)氧化应激相关通路的影响。方法取处于对数生长期的SV-HUC-1细胞,分别暴露于含终浓度为0(对照,F12K培养基)、4μmol/L亚砷酸钠及4μmol/L亚砷酸钠与丁硫氨酸亚砜亚胺(BSO,0.5 mmol/L)、褪黑素(0.5 mmol/L)或N-乙酰半胱氨酸(NAC,0.5 mmol/L)的培养基中,于培养16 h后,收集细胞进行转录因子NF-E2相关因子2(nuclear factor erythroid 2-related factor 2,NRF2)通路相关蛋白及其下游基因[血红素加氧酶1(heme oxygenase-1,HO1)、醌氧化还原酶(NAD(P)H-quinone oxidoreductase 1,NQO1)]蛋白表达水平的检测;于培养1 h后,收集细胞进行丝裂原活化蛋白激酶(mitogen-Activated Protein Kinase,MAPK)通路关键蛋白[p-细胞外信号调节激酶(p-ERK)、p-p38、p-c-Jun-N末端激酶(p-JNK)]表达水平的检测。结果与对照组比较,亚砷酸钠暴露SV-HUC-1细胞内NRF2、NQO1、HO1蛋白及p-ERK、p-p38、p-JNK蛋白的表达水平均较高;巯基耗竭剂BSO与亚砷酸钠联合暴露可加剧砷诱导的SV-HUC-1细胞内NRF2、NQO1、HO1和p-p38、p-JNK蛋白表达水平的升高,而抑制p-ERK的升高,差异均有统计学意义(P0.05)。褪黑素与亚砷酸钠组及亚砷酸钠+NAC组SV-HUC-1细胞内NRF2蛋白的表达水平明显低于对照组和亚砷酸钠组;NQO1蛋白的表达水平仅明显高于对照组;HO1蛋白的表达水平明显高于对照组,而明显低于亚砷酸钠组,差异均有统计学意义(P0.05)。褪黑素+亚砷酸钠组SV-HUC-1细胞内p-ERK、p-p38蛋白的表达水平明显低于对照组和亚砷酸钠组;p-JNK蛋白的表达水平明显高于对照组和亚砷酸钠组,差异均有统计学意义(P0.05)。亚砷酸钠+NAC组SV-HUC-1细胞内p-p38蛋白的表达水平明显低于对照组和亚砷酸钠组;p-JNK蛋白的表达水平仅明显高于对照组;p-ERK蛋白的表达水平明显高于对照组,而明显低于砷暴露组,差异均有统计学意义(P0.05)。结论抗氧化剂能够抑制亚砷酸钠急性暴露导致的SV-HUC-1细胞氧化应激相关通路的活化。     

Involvement of N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) in arsenic biomethylation and its role in arsenic-induced toxicity

Background

In humans, inorganic arsenic (iAs) is metabolized to methylated arsenical species in a multistep process mainly mediated by arsenic (+3 oxidation state) methyltransferase (AS3MT). Among these metabolites is monomethylarsonous acid (MMA^III), the most toxic arsenic species. A recent study in As3mt-knockout mice suggests that unidentified methyltransferases could be involved in alternative iAs methylation pathways. We found that yeast deletion mutants lacking MTQ2 were highly resistant to iAs exposure. The human ortholog of the yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1), encoding a putative methyltransferase.

Objective

We investigated the potential role of N6AMT1 in arsenic-induced toxicity.

Methods

We measured and compared the cytotoxicity induced by arsenicals and their metabolic profiles using inductively coupled plasma–mass spectrometry in UROtsa human urothelial cells with enhanced N6AMT1 expression and UROtsa vector control cells treated with different concentrations of either iAs^III or MMA^III.

Results

N6AMT1 was able to convert MMA^III to the less toxic dimethylarsonic acid (DMA) when overexpressed in UROtsa cells. The enhanced expression of N6AMT1 in UROtsa cells decreased cytotoxicity of both iAs^III and MMA^III. Moreover, N6AMT1 is expressed in many human tissues at variable levels, although at levels lower than those of AS3MT, supporting a potential participation in arsenic metabolism in vivo.

Conclusions

Considering that MMA^III is the most toxic arsenical, our data suggest that N6AMT1 has a significant role in determining susceptibility to arsenic toxicity and carcinogenicity because of its specific activity in methylating MMA^III to DMA and other unknown mechanisms.     

砷暴露对小鼠脾脏炎症反应及Th1/Th2的影响

目的探讨慢性饮水砷暴露对小鼠脾脏的炎症反应及Th1/Th2细胞因子和转录因子表达的影响。方法将30只健康SPF级雌性昆明小鼠根据体重按照随机数字表分为对照组和25、50 mg/L NaAsO_2染毒组,每组10只。采用自由饮水方式染毒,染毒6个月后,测定小鼠脾脏组织中炎症因子IL-12和IL-6的mRNA水平。采用Real-time PCR方法分别检测脾脏Th1和Th2的转录因子T-bet、Gata3和细胞因子Ifn-γ、IL-4的mRNA表达水平。同时用Western blot法观察Nrf2、NQO1和GSTO1/2的蛋白表达水平。结果与对照组比较,随着染毒剂量的上升,各染毒组小鼠脾脏炎症因子IL-12和IL-6的mRNA水平均上升,除IL-12的25 mg/L剂量组外,差异均具有统计学意义(P0.05);各染毒组小鼠脾脏细胞因子Ifn-γ和IL-4的转录水平呈上升趋势;Nrf2及其调控的下游蛋白NQO1和GSTO1/2的表达水平明显增加。结论慢性砷暴露小鼠免疫器官脾脏的炎症相关因子IL-12和IL-6明显增加,同时上调Th1和Th2特异性细胞因子的表达水平,调节CD4~+T淋巴细胞亚群的分化,从而发挥对小鼠脾脏的免疫调节效应。     

亚砷酸钠对SV-HUC-1细胞NRF2通路影响

目的 探讨亚砷酸钠（NaAsO₂）对人膀胱上皮细胞系SV-HUC-1细胞内核因子相关因子2（NRF2）活性的影响。方法 时间效应实验以4 μmol/L NaAsO₂分别处理SV-HUC-1细胞0（对照）、4、8、16、24 h;剂量效应实验设对照组和NaAsO₂处理组（1、2、4、8、10 μmol/L）;Western blot免疫印迹试验检测NRF2通路相关蛋白（NRF2、NQO1和HO1）表达水平。结果 4 μmol/L NaAsO₂处理SV-HUC-1细胞4、8、16、24 h后,与对照组（0.68±0.03）比较,细胞内NRF2蛋白表达水平明显升高,16 h处理组NRF2蛋白表达最高（1.17±0.10）,差异有统计学意义（P<0.05）;随着染砷剂量增加,NRF2、NQO1和HO1蛋白表达水平增强,与对照组NRF2（0.86±0.05）、NQO1（0.68±0.02）、HO1（0.09±0.01）比较,4和8 μmol/L NaAsO₂处理组细胞内NRF2、NQO1、HO1蛋白表达水平分别为（1.12±0.01）和（1.16±0.11）、（0.93±0.01）和（1.15±0.19）、（1.28±0.01）和（1.30±0.02）,均明显升高,差异均有统计学意义（均P< 0.05）。结论 NaAsO₂急性暴露能够活化NRF2通路,引起膀胱上皮细胞适应性抗氧化反应增强。     

The in vitro cytotoxicity of asbestos fibers: I. P388D1 cells

In this study, the cytotoxicity of 13 fibrous samples of known fiber number and dimensions has been established in P388D1 cells. The cells were exposed in vitro to dust concentrations of 10 or 50 micrograms/ml and, after incubation for 24 or 48 hours, any changes in cellular viability, lactate dehydrogenase, and glucosaminidase levels were determined. In general, there was a close association between the reduction in cellular viability and the loss of intracellular enzymes induced by each dust, the chrysotile asbestos samples proving more cytotoxic than the amphiboles. The cytotoxicity of the fibrous dusts was shown to be related to the number of fibers greater than 8 micron in length in the samples.     

Comparative study on the inhibitory effects of antioxidant vitamins and radon on carbon tetrachloride-induced hepatopathy

We have previously reported that radon inhalation activates anti-oxidative functions and inhibits carbon tetrachloride (CCl₄)-induced hepatopathy. It has also been reported that antioxidant vitamins can inhibit CCl₄-induced hepatopathy. In the current study, we examined the comparative efficacy of treatment with radon, ascorbic acid and α-tocopherol on CCl₄-induced hepatopathy. Mice were subjected to intraperitoneal injection of CCl₄ after inhaling approximately 1000 or 2000 Bq/m³ radon for 24 h, or immediately after intraperitoneal injection of ascorbic acid (100, 300, or 500 mg/kg bodyweight) or α-tocopherol (100, 300, or 500 mg/kg bodyweight). We estimated the inhibitory effects on CCl₄-induced hepatopathy based on hepatic function-associated parameters, oxidative damage-associated parameters and histological changes. The results revealed that the therapeutic effects of radon inhalation were almost equivalent to treatment with ascorbic acid at a dose of 500 mg/kg or α-tocopherol at a dose of 300 mg/kg. The activities of superoxide dismutase, catalase, and glutathione peroxidase in the liver were significantly higher in mice exposed to radon than in mice treated with CCl₄ alone. These findings suggest that radon inhalation has an anti-oxidative effect against CCl₄-induced hepatopathy similar to the anti-oxidative effects of ascorbic acid or α-tocopherol due to the induction of anti-oxidative functions.     

多壁碳纳米管致RAW264.7巨噬细胞毒性与氧化损伤研究

目的探讨多壁碳纳米管(MWCNTs)对小鼠巨噬细胞株RAW264.7细胞的体外细胞毒性和氧化损伤作用。方法用DNA钠盐提高MWCNTs的分散度,设4个浓度组(2.5、10、25和100μg/ml)、DNA钠盐溶剂对照组和生理盐水对照组,染毒24h后,用四甲基偶氮唑盐(MTT)方法观察细胞毒性,并根据染毒后细胞的总蛋白(TP)、乳酸脱氢酶(LDH)、一氧化氮(NO)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和丙二醛(MDA)的变化情况分析MWCNTs的细胞毒性和氧化损伤作用。结果25μg/ml和100μg/ml浓度组细胞存活率及细胞毒性和氧化损伤指标均有不同程度的改变,呈一定的浓度-反应关系;随着染毒浓度的升高,细胞和其上清液中TP、LDH、NO和MDA含量随之升高,而GSH和SOD含量随之降低,且呈一定的暴露-反应关系;且与细胞发生融解破碎,间隙加大等形态学改变情况相符。结论MWCNTs对体外培养巨噬细胞株RAW264.7细胞具有细胞毒性和氧化损伤作用,且随剂量的增大而增强。     

Effects of arsenic on human keratinocytes: morphological,physiological, and precursor incorporation studies

Measurement of in vitro percutaneous absorption of As(III) and As(V) by artificial human skin shows a strong affinity of arsenic for the human keratinocytes, with 1-10% of the applied arsenic dose retained by the artificial skin per hour. The inordinate retention of arsenic by the skin is a risk factor for As toxicity. The calculated permeability constant (K(p)) averaged about 4.3 x 10(-5) cm/h for As(V) and 10.1 x 10(-5) cm/h for As(III). A facile calculation suggests that dermal absorption during showering and hand washing can be an important exposure route if the water contains more than 100 microg/L As(III) or As(V). The effects of the absorbed arsenic in artificial skin were evaluated in terms of morphological characteristics, integrity of the cell membrane (by means of lactate dehydrogenase and MTS assays), and rates of DNA, RNA, and protein synthesis estimated by incorporation of radioactive precursors. We found significant morphological changes, cytotoxicity associated with disruption of the cell membrane, and inhibition of DNA and protein syntheses at As(III) exposure doses as low as 10 microg/L.     

Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines

Summary Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. Their proposed protective role in tumor development may prevail especially in the intestinal tract due to direct exposure of intestinal epithelia to these dietary ingredients. We have screened more than 30 flavonoids for their effects on cell proliferation and potential cytotoxicity in the human colon cancer cell lines Caco-2, displaying features of small intestinal epithelial cells, and HT-29, resembling colonic crypt cells. In addition, for selected compounds we assessed whether they induce apoptosis by determining caspase-3 activation. Studies on the dose dependent effects of the flavonoids showed antiproliferative activity of all compounds with EC₅₀ values ranging between 39.7 ± 2.3 μM (baicalein) and 203.6 ± 15.5 μM (diosmin). In almost all cases, growth inhibition by the flavonoids occured in the absence of cytotoxicity. There was no obvious structure-activity relationship in the antiproliferative effects either on basis of the subclasses (i.e., isoflavones, flavones, flavonols, flavanones) or with respect to kind or position of substituents within a class. In a subset of experiments we examined the antiproliferative activities of the most potent compound of each flavonoid subgroup in addition in LLC-PK₁, a renal tubular cell line, and the human breast cancer cell line MCF-7. Out of four flavonols tested, three displayed almost equal antiproliferative activities in all cell lines but fisetin was less potent in MCF-7 cells. The flavanones bavachinin and flavanone inhibited growth of Caco-2 and HT-29 cells with lower EC₅₀ values than that obtained in LLC-PK₁ and MCF-7 cells. The lower susceptibility of LLC-PK₁ and MCF-7 cells towards growth arrest was even more pronounced in the case of the flavone baicalein. Half maximal growth-inhibition in LLC-PK₁ and MCF-7 required 2.5 and 6.6 fold higher concentrations than that needed in the intestinal cell lines. The flavonoids failed to affect apoptosis in LLC-PK₁ and MCF-7, whereas baicalein and myricetin were able to induce apoptosis in HT-29 and Caco-2 cells. In conclusion, flavonoids of the flavone, flavonol, flavanone, and isoflavone classes possess antiproliferative effects in different cancer cell lines. The capability of flavonoids for growth inhibition and induction of apoptosis can not be predicted on the basis of their chemical composition and structure. Received: 28 December 1998, Accepted: 18 March 1999     

Anti-inflammatory and antioxidant effects of umbelliferone in chronic alcohol-fed rats

BACKGROUND/OBJECTIVESInflammation is associated with various types of acute and chronic alcohol liver diseases. In this study, we examined whether umbelliferone (7-hydroxycoumarin, UF) ameliorates chronic alcohol-induced liver damage by modulating inflammatory response and the antioxidant system.METHODSRats were fed a Liber-Decarli liquid diet containing 5% alcohol with or without UF (0.05 g/L) for 8 weeks, while normal rats received an isocaloric carbohydrate liquid diet.RESULTSChronic alcohol intake significantly increased serum tumor necrosis factor-α (TNF-α) and interleukin 6 levels and decreased interleukin 10 level; however, UF supplementation reversed the cytokines related to liver damage. UF significantly suppressed hepatic lipopolysaccharide binding protein, toll-like receptor 4 (TLR4), nuclear factor kappa B, and TNF-α gene expression increases in response to chronic alcohol intake. Masson''s trichrome staining revealed that UF improved mild hepatic fibrosis caused by alcohol, and UF also significantly increased the mRNA expressions and activities of superoxide dismutase and catalase in liver, and thus, decreased lipid peroxide and mitochondrial hydrogen peroxide levels.CONCLUSIONSThe findings of this study indicate that UF protects against alcohol-induced liver damage by inhibiting the TLR4 signaling pathway and activating the antioxidant system.     

The effects of cranberry juice consumption on antioxidant status and biomarkers relating to heart disease and cancer in healthy human volunteers

Summary Background Consumption of fruit and vegetables is associated with a decreased risk of heart disease and cancer.This has been ascribed in part to antioxidants in these foods inactivating reactive oxygen species involved in initiation or progression of these diseases. Non–nutritive anthocyanins are present in significant amounts in the human diet. However, it is unclear whether they have health benefits in humans. AimTo determine whether daily consumption of anthocyanin–rich cranberry juice could alter plasma antioxidant activity and biomarkers of oxidative stress. Methods 20 healthy female volunteers aged 18–40 y were recruited. Subjects consumed 750 ml/day of either cranberry juice or a placebo drink for 2 weeks. Fasted blood and urine samples were obtained over 4 weeks.The total phenol, anthocyanin and catechin content of the supplements and plasma were measured. Anthocyanin glycosides were identified by tandem mass spectrometry (MS–MS). Vitamin C, homocysteine (tHcy) and reduced glutathione (GSH) were measured by HPLC. Total antioxidant ability was determined using electron spin resonance (ESR) spectrometry and by the FRAP assay. Plasma total cholesterol, high density lipoprotein (HDL), and low density lipoprotein (LDL) cholesterol and triglycerides (TG) were measured. Glutathione peroxidase (GSH–Px), catalase (CAT) and superoxide dismutase (SOD) activities were measured in erythrocytes. Urine was collected for analysis of malondialdehyde (MDA) by HPLC and 8–oxo–deoxyguanosine (8–oxo–dG) by ELISA.Endogenous and induced DNA damage were measured by single cell gel electrophoresis (SCGE) in lymphocytes. Results Vitamin C, total phenol, anthocyanin and catechin concentrations and FRAP and ESR values were significantly higher in the cranberry juice compared with the placebo. Cyanidin and peonidin glycosides comprised the major anthocyanin metabolites [peonidin galactoside (29.2%) > cyanidin arabinoside (26.1%) > cyanidin galactoside (21.7%) > peonidin arabinoside (17.5%) > peonidin glucoside (4.1%) > cyanidin glucoside (1.4 %)]. Plasma vitamin C increased significantly (P<0.01) in volunteers consuming cranberry juice. No anthocyanins (plasma) or catechins (plasma or urine) were detectable and plasma total phenols, tHcy,TC,TG,HDL and LDL were unchanged. The antioxidant potential of the plasma, GSH–Px, CAT and SOD activities, and MDA were similar for both groups. Supplementation with cranberry juice did not affect 8–oxo–deoxyguanosine in urine or endogenous or H₂O₂–induced DNA damage in lymphocytes. Conclusions Cranberry juice consumption did not alter blood or cellular antioxidant status or several biomarkers of lipid status pertinent to heart disease. Similarly, cranberry juice had no effect on basal or induced oxidative DNA damage.These results show the importance of distinguishing between the in vitro and in vivo antioxidant activities of dietary anthocyanins in relation to human health.     

Comparative effects of dietary flavanols on antioxidant defences and their response to oxidant-induced stress on Caco2 cells

Purpose  

Flavanols are an important fraction of our diet both for their antioxidant capacity and because they are constituents of greatly accepted foodstuffs such as tea, wine and cocoa. In addition to their antioxidant activity by directly scavenging intracellular reactive oxygen species (ROS), flavanols have been recently shown to enhance protective enzymes. The objective was to evaluate the antioxidant response of colon-derived Caco2 cells to dietary flavanols.     

3种抗氧化剂对亚砷酸钠染毒人膀胱上皮细胞血管内皮生长因子表达的拮抗作用研究

目的探讨抗氧化剂褪黑素(melatonin,MEL)、N-乙酰半胱氨酸(N-acetyl cysteine,NAC)、维生素C(Vitamin,VC)对砷诱导人正常膀胱上皮(SV-HUC-1)细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的拮抗作用。方法将处于对数生长期的SV-HUC-1细胞暴露于含终浓度分别为0(对照)、0.5、1、2、4、8、10μmol/L亚砷酸钠的F12K完全培养基染毒24 h,或者含终浓度分别为4、10μmol/L亚砷酸钠的F12K完全培养基染毒0(对照)、4、12、24、48、72 h;联合暴露组在加入含终浓度分为4μmol/L亚砷酸钠的F12K完全培养基前30 min分别加入1%二甲基亚砜(DMSO)和抗氧化剂MEL、VC、NAC(终浓度分别为0.5、1、1 mmol/L),染毒24 h。分别采用Western blot法和RT-PCR法检测SV-HUC-1细胞VEGF蛋白和mRNA的表达水平。结果 10μmol/L亚砷酸钠染毒组SV-HUC-1细胞VEGF mRNA的表达水平高于对照组,差异有统计学意义(P0.05);且随着亚砷酸钠染毒剂量的升高,SV-HUC-1细胞VEGF mRNA的表达水平呈上升趋势。与对照组比较,4μmol/L亚砷酸钠染毒12、24、48 h及10μmol/L亚砷酸钠染毒24和48 h后SV-HUC-1细胞VEGF mRNA的表达水平均升高,差异有统计学意义(P0.05);且随着亚砷酸钠染毒时间的延长,各剂量组SV-HUC-1细胞VEGF mRNA的表达水平均呈先上升后下降的趋势。与对照组比较,4μmol/L亚砷酸钠+DMSO染毒组SV-HUC-1细胞中VEGF蛋白的表达水平均增加,差异有统计学意义(P0.05)。与4μmol/L亚砷酸钠+DMSO染毒组比较,亚砷酸钠+NAC染毒组SV-HUC-1细胞中VEGF蛋白的表达水平较高,差异有统计学意义(P0.05);而亚砷酸钠与MEL和VC联合染毒组SV-HUC-1细胞中VEGF蛋白的表达水平无明显改变。结论砷能诱导人正常膀胱上皮细胞VEGF表达增加,NAC能增加VEGF表达。     

PKRP、TURP治疗前列腺增生疗效及对ET-1、PGE2和IL-6的影响

目的探讨经尿道等离子双极电切术(PKRP)、经尿道前列腺电切术(TURP)治疗前列腺增生疗效及对内皮素-1 (ET-1)、前列腺素E2(PGE2)、白介素-6(IL-6)的影响。方法分析2018年4月至2020年4月安阳市人民医院收治的122例前列腺增生患者,根据不同治疗方式分为研究组(PKRP治疗,n=63)和对照组(TURP治疗,n=59)。比较两组临床疗效、手术相关情况、ET-1、PGE2、IL-6水平及并发症发生率。结果研究组总有效率(92.06%)与对照组(94.92%)比较,差异无统计学意义(P>0.05)。研究组手术时长、术中出血量、住院时间少于对照组,差异有统计学意义(P<0.05)。与术前相比,术后两组ET-1、PGE2、IL-6水平均上升,但对照组ET-1、PGE2、IL-6水平较研究组上升更为明显,差异有统计学意义(P<0.05)。研究组术后并发症总发生率(6.35%)明显低于对照组(20.34%),差异有统计学意义(P<0.05)。结论 PKRP与TURP治疗前列腺增生的临床疗效相当,但PKRP治疗创伤更小,术后恢复快,对ET-1、PGE2、IL-6的影响更小,且安全性较高,值得临床应用推广。