苯并 (a)芘染毒致人支气管上皮细胞和上皮样成纤维细胞周期的变化

目的： 观察苯并 (a)芘[benzo (a)pyrene,BaP]染毒对人支气管上皮细胞 (16HBE)和上皮样肺成纤维细胞 (W138)细胞周期的影响,并探讨其剂量效应和时间效应。方法：以16HBE和W138细胞未处理组为阴性对照组,二甲基亚砜 (DMSO)组为溶剂对照组。分别以不同浓度 (1、2、4、8、16、32 μmol/L)的BaP染毒16HBE和W138细胞,24 h后用流式细胞术检测细胞周期分布情况;分别以16 μmol/L BaP染毒16HBE和W138细胞,作用不同时间后 (1、2、4、8、12、24 h)检测细胞周期分布情况;分别以16 μmol/L BaP染毒16HBE和W138细胞,作用4 h后,再经过不同时段 (0、1、2、4、8、12、24 h)的恢复期后检测细胞周期分布情况。结果：与阴性对照组比较,随着染毒浓度和染毒时间的增加16HBE和W138 细胞S期所占比例均增加 (P<0.05);16 μmol/L BaP染毒16HBE细胞4 h后,经2~12 h的恢复期 (正常条件下培养),S期细胞所占比例与刚染毒后细胞 (32.43%)相比明显增加 (P<0.05),恢复24 h时S期细胞所占比例 (24.52%)与阴性对照组 (26.41%)相比差异无统计学意义 (P>0.05);16 μmol/L BaP染毒W138细胞4 h后恢复0~4 h时S期细胞所占比例与阴性对照组 (32.42%)相比明显增加 (P<0.05),恢复8 h开始减少,24 h时S期细胞所占比例 (32.89%)与阴性对照组 (32.42%)相比差异无统计学意义 (P>0.05)。结论： BaP染毒16HBE和W138细胞引起细胞周期分布变化,主要为S期阻滞,G1期和G2期变化不明显。     

泛素连接酶RING2对苯并[a]芘染毒人支气管上皮细胞周期和P53蛋白表达的影响

目的:探讨泛素连接酶RING2对苯并[a]芘(BaP)染毒人支气管上皮16HBE细胞周期和P53蛋白表达的影响。方法:以16HBE未处理组为阴性对照组,二甲基亚砜(DMSO)组为溶剂对照组,MOCK组为序列对照组。在使用RNA干扰技术降低16HBE细胞泛素连接酶RING2基因表达前后,分别采用不同浓度BaP(1、2、4、8、16、32 μmol/L)染毒24 h;或16 μmol/L BaP染毒不同时间(1、2、4、8、12、24 h)。用流式细胞术检测干扰前后两组细胞周期分布情况,用Western-blot法检测干扰前后两组细胞P53蛋白表达水平。结果:流式细胞术检测结果显示,与阴性对照组比较,16HBE细胞染毒后各浓度和各时点组S期细胞所占的比例均增加(P<0.05),而16HBE(siRNA-RING2)各浓度和各时点组S期细胞所占的比例均下降(P<0.05)。协方差分析显示分组因素(是否进行RNAi)和染毒浓度都对S期细胞比例有影响(P均<0.01),16HBE(siRNA-RING2)细胞组的修正均数(17.09%)比16HBE细胞组(31.55%)明显降低(P<0.01)。分组因素和染毒时间都对S期细胞比例有影响(P均<0.01),16HBE(siRNA-RING2)细胞组的修正均数(13.07%)比16HBE细胞组(28.04%)明显下降(P<0.01)。Western-blot结果显示,与阴性对照组比较,16HBE 细胞染毒后各浓度和各时点组P53的表达水平均增加(P<0.05),而16HBE(siRNA-RING2)细胞除16 μmol/L染毒8 h组外,其余各组P53的表达水平均降低(P<0.05)。协方差分析显示分组因素和染毒浓度都对P53的表达水平有影响,P值分别为0.026和0.028。16HBE(siRNA-RING2)细胞组的修正均数(0.989)比16HBE细胞组(1.375)明显下降(P<0.05);分组因素 和染毒时间都对P53的表达水平有影响,P值分别为0.007和0.035。16HBE(siRNA-RING2)细胞组的修正均数(0.857)比16HBE细胞组(1.541)明显下降(P<0.05)。结论:RING2参与的组蛋白泛素化修饰可能通过影响 P53表达和细胞周期S期的变化来发挥对DNA损伤修复的调控。     

长期高剂量苯并(a)芘染毒对小鼠肺脏microRNAs表达的影响

目的:研究长期高剂量苯并(a)芘[B(a)P染毒对小鼠肺组织miRNAs表达谱的影响,探讨miRNAs在B(a)P健康损害过程中的作用。方法:40只ICR小鼠随机分为对照组和染毒组,每组20只,雌雄各半。经口灌胃给予小鼠50 mg/kg B(a)P,每周2次,持续8周,对照组同时给予等量的橄榄油,染毒结束后继续饲养8周。取肺脏组织,提取总RNA,采用SOliD高通量测序技术检测miRNAs表达谱,进行miRNAs差异表达分析,并用real time-PCR验证miRNAs表达,TargetScan,miRanda及picTar软件预测miRNAs可能调控的靶基因。结果:绝大部分miRNAs在肺组织的表达信号强度较低。与对照组相比,B(a)P染毒组小鼠肺组织中共有109个miRNAs发生差异表达,其中50个miRNAs表达上调,59个miRNAs表达下调。上调幅度最大的为7.43倍,下调幅度最大的为40.63倍。为验证测序的结果,取下调较为明显的miR-20b生行real time-PCR分析,结果显示与测序结果相一致,靶基因预测显示miR-20b可能调节与细胞增殖、周期、凋亡及肿瘤发生相关的蛋白。结论:长期高剂量B(a)P染毒可引起小鼠肺组织miRNAs表达产生特异性改变,差异表达miRNAs可能在B(a)P致机体健康损害过程中起着重要作用。     

苯并a芘对人胎气管上皮细胞生物大分子作用的探讨

苯并ａ芘（ＢａＰ）是烧煤和吸烟过程中最常见的化学污染物，为了解其在人类肺癌发生中的作用，用人胎肝肺组织和气管支气管上皮细胞（ＨＦＢＥ）研究了ＢａＰ的代谢、几种主要代谢物的致微核（ＭＮ）作用、对程序外ＤＮＡ合成（ＵＤＳ）的影响和对Ｈ－ｒａｓ癌基因的致突变作用。结果发现人胎肝肺组织的微粒体酶都有代谢ＢａＰ的能力，初级代谢产物中有三种二氢二醇苯并ａ芘的衍生物，两种羟基苯并ａ芘代谢物和一种醌基苯并ａ芘。用于试验的ＢａＰ的五种主要代谢物均可诱发ＨＦＢＥ的ＵＤＳ，其中以ａｎｔｉ－ＢＰＤＥ和７．８－ｄｉｏｌＢａＰ最为明显；有三种代谢物可致微核率升高，也是ａｎｔｉ－ＢＰＤＥ的作用最强。用ＰＣＲ－ＲＦＬＰ法发现ａｎｔｉ－ＢＰＤＥ处理后，ＨＦＢＥ中Ｈ－ｒａｓ癌基因的第１２位密码子发生了点突变。本研究对Ｂａｐ的致肺癌作用的可能机制从分子水平上提供了进一步的资料。     

苯并(a)芘对褐菖肝脏DNA损伤与抗氧活性的影响

背景与目的:研究苯并(a)芘BaP对褐菖鲉的毒性效应.材料与方法:将褐菖纳分别暴露于不同浓度(10、100、1 000 ng/L)的苯并(a)芘,0、7、25和50 d以及恢复期7、20 d取鱼肝脏,测定超氧化物歧化酶(SOD)、谷胱甘肽硫转移酶(GST)活性,还原型谷胱甘肽(GSH)含量和DNA单链断裂指标.实验同设溶剂对照组.结果:总SOD活性在Bap暴露7 d后被抑制,25 d后,10 ng/L和100ng/L Bap组SOD活性升高(P<0.05);50 d时,1 000 ng/L组SOD活性显著升高(P<0.05).10 ng/L BaP暴露7 d以及100 ng/L和1 000 ng/L BaP暴露50 d时,GSH含量显著增加(P<0.05).而GST活性在100 ng/L和1 000 ng/L BaP分别暴露25 d、50 d时显著增加,随着暴露时间的延长和暴露浓度的增加,各BaP浓度组DNA损伤呈加重趋势.结论:褐菖鲉肝脏SOD、GST酶活性与GSH含量结合使用以及DNA单链断裂损伤可以作为监测海洋环境中多环芳烃(PAHs)污染的潜在生物标志物.     

多氯联苯126与苯并(a)芘联合作用致代谢酶改变对HepG2细胞遗传毒性的影响

背景与目的:探讨多氯联苯126(PCB126)对苯并(a)芘[B(a)P]遗传毒性的影响.材料与方法:设PCB126三个剂量(0.01、0.10、1.00 nmol/L),B(a)P一个剂量(50 μmol/L)组,设三甲基胆蒽(3-MC)为阳性对照,二甲基亚砜(DMSO)为溶剂对照,以各PCB126浓度染毒HepG2细胞48 h后,再与B(a)P联合染毒24h.通过荧光分光光度法测定各组细胞CYP1A1酶活性(EROD);并采用胞质分裂阻滞法微核实验(CBMNT)分析各组细胞的微核率(MN%o),并计算核分裂指数(NDI).结果:与溶剂对照相比,PCB126各浓度组和50 μmol/L的B(a)P单独作用及联合作用均可诱导CYP1A1酶活性显著增加,其差异均具有统计学意义(P＜0.05,P＜0.01).微核率显著升高仅见于50 μmol/L的B(a)P单独作用组,与溶剂对照组相比差异有统计学意义(P＜0.01).0.10、1.00 nmol/L的PCB126和50 μmol/L的B(a)P联合作用时,与B(a)P单独作用相比,CYP1A1酶活性和微核率均显著升高,差异有统计学意义(P＜0.05,P＜0.01).结论:PCB126在本试验条件下未显示出遗传毒性作用,但对B(a)P的遗传毒性作用具有一定的增强效应.     

Aroclor1254预先染毒增强苯并[a]芘对Hep G2细胞DNA的损伤

目的: 探讨Hep G2细胞经Aroclor1254预处理后对苯并[a]芘诱发的DNA损伤的影响. 方法: 运用单细胞凝胶电泳技术检测了Hep G2细胞经Aroclor1254(23、46、92和184 μmol / L)单独染毒24 h和将其预处理24 h后再用苯并[a]芘染毒1 h对DNA损伤的影响. 结果: 苯并[a]芘诱发的DNA损伤随着Aroclor1254预处理的浓度增大而升高,呈明显的剂量效应关系.当Aroclor1254预处理的浓度分别为46、92和184 μmol / L时,苯并[a]芘诱发的DNA损伤与苯并[a]芘单独作用相比分别升高了8 %、16 %和160 %.184 μmol / L的Aroclor1254预处理后,苯并[a]芘诱发的DNA损伤与苯并[a]芘单独作用相比有极显著性差异(P<0.01). 结论: Aroclor1254可显著地增强苯并[a]芘在Hep G2细胞中诱导的DNA损伤,这种损伤的增强可能和Aroclor1254对Hep G2细胞CYP1A1的诱导有关.     

p63和p73的表达与苯并(a)芘致H1299和16HBE细胞DNA损伤的关系

背景与目的:研究p63与p73的mRNA表达与BaP致人肺腺癌细胞(H1299)和人支气管上皮细胞(16HBE)DNA损伤的关系.材料与方法:分别用不同浓度BaP(8、16、32、64和128 μmol/L)处理H1299和16HBE两种细胞,在4 h和12 h时,使用相应的生化检测试剂盒分别测定细胞裂解液中MDA的水平和SOD、GSH-Px的活性,用qRT-PCR方法测定处理后细胞的p53、p63、p73、mdm2和mdm4的mRNA水平;用Comet实验评价细胞DNA损伤程度.结果:16、32和64 μmol/L BaP处理4 h时,两种细胞MDA水平显著性升高,SOD和GSH-Px活性显著性下降(P<0.05).用BaP处理H1299和16HBE细胞4 h和12 h时均观察到DNA损伤随浓度增加而加重,且呈剂量-效应关系(P<0.01),mdm2、mdm4 mRNA表达水平升高(P<0.01).不过仅在12 h时p53基因mRNA表达水平较对照组显著增加(P<0.01).在4 h和12 h时点,仅在H1299细胞的p63和p73 mRNA表达增加(P<0.05). 结论:在BaP致p53缺失的H1299细胞的DNA损伤中,BaP可能通过不依赖p53信号通路激活了p63和p73 mRNA的表达.     

Poly(ADP-ribose) glycohydrolase silencing down-regulates TCTP and Cofilin-1 associated with metastasis in benzo(a)pyrene carcinogenesis

Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant. BaP is a known carcinogen and can induce malignant transformation of rodent and human cells. Many evidences suggest that inhibitor of poly(ADP-ribose) glycohydrolase (PARG) is potent anticancer drug candidate. However, the effect of PARG on BaP carcinogenesis remains unclear. We explored this question in a PARG-deficient human bronchial epithelial cell line (shPARG cells) treated with various concentration of BaP for 15 weeks. Soft agar assay was used to examine BaP-induced cell malignancy of human bronchial epithelial cells and shPARG cells. Mechanistic investigations were used by 2D-DIGE and mass spectrometry. Western blot analysis and Double immunofluorescence detection were used to confirm some of the results obtained from DIGE experiments. We found that PARG silencing could dramatically inhibit BaP-induced cell malignancy of human bronchial epithelial cells in soft agar assay. Altered levels of expression induced by BaP were observed within shPARG cells for numerous proteins, including proteins required for cell mobility, stress response, DNA repair and cell proliferation pathways. Among these proteins, TCTP and Cofilin-1 involved in malignancy, were validated by western blot analysis and immunofluorescence assay. PARG inhibition contributed to down-regulation of TCTP and Cofilin-1. This is the first experimental demonstration of a link between PARG silencing and reduced cell migration after BaP exposure. We propose that PARG silencing might down-regulate TCTP and Cofilin-1 associated with metastasis in BaP carcinogenesis.     

苯并（a）芘和滴滴涕联合暴露对小鼠肝脏细胞凋亡的影响

目的：探讨不同剂量苯并（a）芘[benzo（a）pyrene,B（a）P]、滴滴涕（chlorophenothane,DDT）单独及联合暴露对小鼠肝脏细胞的毒性效应。方法：成年雌性昆明种小鼠66只,随机分为11组：分别为0.5、5、50mg/（kg·d）B（a）P染毒组,0.025、0.25、2.5mg/（kg·d）DDT染毒组,0.5mg/（kg·d）B（a）P＋0.025mg/（kg·d）DDT联合染毒组,5mg/（kg·d）B（a）P＋0.25mg/（kg·d）DDT联合染毒组,50mg/（kg·d）B（a）P＋2.5mg/（kg·d）DDT联合染毒组,空白对照组（正常饲养）和溶剂对照组（植物油处理）。染毒组用含B（a）P、DDT的食用油进行腹腔注射,每天1次,连续21d,于末次给药24h后处死小鼠。取肝脏制作冰冻切片,利用原位缺口未端标记（terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL）法检测肝细胞凋亡情况。结果：5和50mg/（kg·d）B（a）P染毒组小鼠肝脏细胞凋亡率显著高于对照组（P〈0.05）,且50mg/（kg·d）剂量组显著高于0.5和5mg/（kg·d）剂量组（P〈0.05）;各DDT染毒组与对照组比较,差异无统计学意义（P〉0.05）;各浓度联合染毒组小鼠肝脏细胞凋亡率均高于对照组（P〈0.01）,各联合染毒组细胞凋亡率间的差异无统计学意义（P〉0.05）,联合染毒组细胞凋亡率和相应的B（a）P染毒组比较,差异无统计学意义（P〉0.05）。结论：B（a）P的单独暴露以及与DDT的联合暴露均可导致小鼠肝脏细胞凋亡的发生,并可能引发其他毒性效应。     

Induction of epithelial-mesenchymal transition-related genes by benzo[a]pyrene in lung cancer cells

BACKGROUND: It is believed that epithelial-mesenchymal transition (EMT) occurs during the development and progression of cancer; however, the correlation between tobacco smoking and EMT remains to be elucidated. METHODS: Cells from the bronchioloalveolar carcinoma cell line A549 were exposed to benzo(a)pyrene (B[a]P) for 24 weeks, and morphology, proliferative activity, and gene expression profiles were analyzed. RESULTS: Although no apparent morphologic changes were observed, the B[a]P-exposed A549 cells exhibited enhanced proliferative activity in 1% bovine serum that contained medium, and dramatic changes in expression levels were observed in a large number of genes. Of those, the expression of EMT-related genes, such as migration-stimulating factor, plasminogen activator inhibitor-1, fibronectin, twist, transforming growth factor-beta2, basic fibroblast growth factor, and electron transport system, were up-regulated; whereas gene expression of E-cadherin was decreased. Most enhanced expression levels remained 8 weeks after the retrieval of B[a]P in culture. CONCLUSIONS: The current results indicated that B[a]P seems to induce EMT in lung cancer cells, and it also may drive disease progression in patients with lung cancer.     

视黄酸对致癌剂苯芘环氧化及其与DNA结合的抑制作用

本文采用氚标记苯芘及体外细胞培养方法,探讨维生素A的重要衍生物视黄酸的防癌机理,发现视黄酸对前致癌剂苯芘环氧化为终致癌剂及其与BALB/c系小鼠骨髓瘤细胞DNA的结合均有显著的抑制作用(P<0.01)。     

L－519，A PHENOLIC COMPOUND，INHIBITS METABOLISM OF BENZO（a）PYRENE AND MUTAGENESIS INDUCED BY BENZO（a）PYRENE

Ｌ－５１９，ＡＰＨＥＮＯＬＩＣＣＯＭＰＯＵＮＤ，ＩＮＨＩＢＩＴＳＭＥＴＡＢＯＬＩＳＭＯＦＢＥＮＺＯ（ａ）ＰＹＲＥＮＥＡＮＤＭＵＴＡＧＥＮＥＳＩＳＩＮＤＵＣＥＤＢＹＢＥＮＺＯ（ａ）ＰＹＲＥＮＥ￥ＣｈｅｎＸｉａｏｇｕａｎｇ；陈晓光；ＦｕＺｈａｏｄｉ；付招...     

The role of 12 cDNA-expressed human,rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7, 8-dihydrodiol

The potent carcinogen benzo[a]pyrene (B[a]P) and its metabolite B[a]P trans-7, 8-dihydrodiol (7, 8-diol) require metabolic activation by the microsomal cytochrome P450s (P450s) to exert several adverse biological effects, including binding to DNA, toxicity, mutagenicity, and carcinogenicity. In the study reported here, we defined the role of each of 12 individual cDNA-expressed cytochrome P450s in the metabolism of B[a]P and 7, 8-diol. Human P450s 1A1 and 1A2 were expressed in the absence or presence of epoxide hydrolase (EH) in a human lymphoblastoid cell line, and six human and five rodent and rabbit P450s were expressed from cDNA with vaccinia virus vectors in the hepatoma cell line Hep G2. B[a]P metabolism resulted in nine metabolites (three diols, three quinones, and three phenols), which were separated, identified, and quantitated by high-pressure liquid chromatography. In the human lymphoblastoid cells, human 1A1 metabolized B[a]P at a rate 4.5 times greater than that for 1A2. EH was shown to be directly involved in B[a]P activation, since increasing the amount of EH resulted in less 7-hydroxybenzo[a]pyrene and more 7, 8-diol formation. Of the human P450s expressed with the vaccinia virus vectors in Hep G2 cells, 1A2 and 2C9 showed the highest activity and 2B6 showed moderate activity for B[a]P metabolism. Mouse 1A1 had activity 40 times higher than any human, rabbit, or rodent P450s, indicating the potential pitfalls of extrapolating P450 activity across species. Metabolism of the 7, 8-diol resulted in six metabolites (four tetrols and two triols). In the lymphoblastoid cells, human 1A1 was shown to be 4.2 times more active than 1A2 for 7, 8-diol metabolism. Among human P450s expressed from vaccinia virus, 1A2, 2E1, and 2C9 gave the highest activity, and 2C8 and 3A4 showed moderate activity for 7, 8-diol metabolism to the diol epoxides. Again, mouse 1A1 was much more active than any other P450. These studies, in which we determined the capacity of individual P450 in the metabolism and activation of B[a]P and 7, 8-diol, may thus lead to a better understanding of how P450s control the detoxification and activation of polycyclic aromatic hydrocarbons. © 1994 Wiley-Liss, Inc.     

PM2.5对支气管上皮细胞DNA损伤作用研究

目的：探讨大气细颗粒物（PM_2.5）染毒对人支气管上皮细胞（HBE） DNA损伤的作用。方法：分别用8、20、50 μg/mL的PM_2.5水溶液染毒HBE细胞24 h后,单细胞凝胶电泳实验（SCGE）检测DNA损伤情况。10和50 μg/L的PM_2.5水溶液染毒HBE细胞,以未染毒细胞作为阴性对照组,10 μmol/L的Cr⁶⁺水溶液为阳性对照组,实时荧光定量PCR （qPCR）检测DNA损伤修复基因hOGG1、hMTH1的mRNA表达水平的变化,Western blot检测hOGG1、hMTH1蛋白表达变化。结果：单细胞凝胶电泳检测8、20和50 μg/mL PM_2.5水溶液染毒组HBE细胞的尾部DNA含量、尾长、尾距较阴性对照组明显增加（P < 0.05或P < 0.01）。qPCR结果显示,与阴性对照组比较,HBE细胞hOGG1 mRNA表达水平在10和50 μg/mL PM_2.5水溶液染毒以及阳性对照Cr⁶⁺水溶液染毒后分别升高75.0%、132.0%、214.0%;hMTH1 mRNA分别升高61.0%、144.0%、75.0%。Western blot结果显示,与阴性对照组比较,HBE细胞hOGG1蛋白表达水平在10和50 μg/mL PM_2.5水溶液染毒以及Cr⁶⁺水溶液染毒后分别升高47.6%、64.0%、47.0%;hMTH1蛋白分别升高20.5%、49.8%、20.9%。结论：PM_2.5水溶液染毒对HBE细胞DNA具有明显的损伤作用,并引起HBE细胞DNA损伤修复基因hOGG1、hMTH1表达水平升高。