Exposure to urban and rural air pollution: DNA and protein adducts and effect of glutathione-S-transferase genotype on adduct levels

Ambient air in urban areas is polluted by agents suspected of causing cancer in humans. A number of epidemiological studies have revealed an increased cancer risk in urban communities, especially in lung cancer. The relative risk have been estimated to be in the order of 1.5. The objective of this study was to evaluate differences in genotoxic exposure through air pollution in urban and rural areas using DNA and protein adducts as biomarkers. Another objective was to investigate whether the GSTM1 genotype has any effect on adduct level. The analyses included ³²P postlabelling of DNA adducts in lymphocytes, enzyme-linked immunosorbent assay for measuring benzo[a]pyrene protein adducts and polymerase chain reaction amplification of the GSTM1 genotype. The study was a cross-sectional study of non-smoking, healthy males from rural and urban Danish areas and from Athens, Greece. All individuals in the study were healthy, non-smoking males. The Danish urban group included 74 university students, the rural group 29 students from agricultural colleges and the Greek group 17 individuals. Adduct levels differed significantly in the three groups with median levels of 0.152 fmol/g DNA (rural), 0.205 fmol/g (urban) and 0.285 fmol/g (Athens). The adduct patterns showed some identical spots, but also specific adducts. Here we report increasing DNA adduct levels comparing residents in rural, small urban and large urban residential areas; we found no influence of GSTM1 genotype on DNA or protein adduct levels in non-smokers exposed to low levels of air pollution.     

GSTM1和GSTM3基因多态性在广东省部分地区健康人群中分布及其基因连锁分析

目的 了解谷胱甘肽-S-转移酶M1和M3(GSTM1、GSTM3)基因多态性在目标人群中分布规律,探索两基因分布之间的联系及其与人群家族史之间的相关关系.方法 应用聚合酶链式反应-2%琼脂糖凝胶电泳、聚合酶链式反应-12%聚丙烯酰胺凝胶电泳的方法检测广东省部分地区606例调查对象的GSTM1、GSTM3基因型.结果 在调查人群中GSTM1(-)基因型的出现频率为56.8%(339/597);调查对象中GSTM3野生纯合子*A-*A、杂合子*A-*B、突变纯合子*B-*B出现的频率分别为62.3%(335/570)、25.8%(147/570)、11.9%(681/570).GSTM1和GSTM3不同基因型在人群中的联合表达有统计学意义(P＜0.05).有冠心病家族史的个体具有GSTM3*B-*B基因型的概率较低(P＜0.05).结论 GSTM1与GSTM3基因在被调查人群中分布有连锁现象;人群中GSTM3*B-*B基因型的表达与人群冠心病家族史之间有统计学关联.     

Ambient and biochemical effect monitoring of workers exposed to ethylene oxide

Objectives: Ethylene oxide is an alkylating agent known to be a directly acting mutagen and carcinogen. This study describes the relationship between workplace ambient air concentrations of ethylene oxide and the concentration of N-2-hydroxyethylvaline in the globin of exposed workers. Methods: During the sterilization of medical equipment, 12 workers were occupationally exposed to ethylene oxide. Personal and stationary ambient air measurements were carried out to monitor the external exposure. The determination of the protein adducts was based on the N-alkyl-Edman method, introducing a new commercially available dipeptide standard for calibration purposes. Results: Ethylene oxide concentrations ranging from 0.2 to 8.5 ppm were found in the workplace air. The adduct concentrations ranged from 5,219 to 32,738 pmol N-2-hydroxyethylvaline/g globin in the case of regularly exposed workers (n = 9) and from 518 to 3,321 pmol N-2-hydroxyethylvaline/g globin for three persons with occasional contact with ethylene oxide. Conclusions: The Deutsche Forschungsgemeinschaft established in 1993 a relationship between the ethylene oxide concentration in ambient air and the amount of N-2-hydroxyethylvaline in human globin. By extrapolation, constant exposure to 1 ppm ethylene oxide should yield approximately 4,000 pmol N-2-hydroxyethylvaline/g globin. The ambient air concentrations of ethylene oxide and the amount of N-2-hydroxyethylvaline determined within the present study confirm this extrapolation in practice. In addition, the determination of adducts based on the use of commercially available dipeptide standards for calibration purposes turned out to be an advantageous alternative to the commonly used protein standards. Received: 18 February 1997 / Accepted: 9 June 1997     

苯DNA加合物的形成特征,条件和方法探讨

为进一步探讨苯ＤＮＡ加合物的形成特征，稳定地检测苯ＤＮＡ加合物，采用３２Ｐ－后标记法对染苯小鼠体内ＤＮＡ加合物的形成特征及影响因素进行了研究。结果表明：加合物种类与染苯天数有关：５００ｍｇ／ｋｇ染苯５天组外周血白细胞可检出２个ＤＮＡ加合物斑点，而７天组可检出３个主要的和至少１个微弱的加合物斑点；染苯达一定剂量后，加合物含量和种类不再增加，１０００ｍｇ／ｋｇ与５００ｍｇ／ｋｇ染苯７天组的检测结果基本相同；苯醌可能是苯形成ＤＮＡ加合物的主要代谢产物，２．５ｍｇ／ｋｇ苯醌染毒５天的小鼠白细胞中可检出３个主要的及２个微弱的加合物斑点，其层析特性与染苯小鼠加合物的层析性质基本一致。提示：动物染苯天数及次数、层析点样量及层析时间对加合物斑点形成均有影响。     

中国南方客家人群谷胱甘肽硫转移酶A1基因多态性研究

目的了解谷胱甘肽硫转移酶A1(GSTA1)基因多态性在中国南方客家人群中的分布规律。方法采用聚合酶链式反应-限制性酶切片段长度多态性(PCR-RFLP)方法检测广东省梅州地区480名客家健康居民GSTA1基因型,应用SPSS10·0统计软件进行统计分析。结果调查人群GSTA1*A/*A、GSTA1*A/*B、GSTA1*B/*B三种基因型的分布频率分别为77·1%、21·7%和1·2%,三种基因型的分布符合哈迪-温伯格(Hardy-Weinberg)平衡吻合度定律。在不同年龄、性别、生活习惯人群中分布差异无显著性,二分类Logistic回归分析结果显示,高血压、冠心病、脑卒中、肺癌及鼻咽癌等家族史在人群中的发生与人群GSTA1不同基因型的分布无显著相关关系。结论中国南方客家人群GSTA1基因型分布呈现多态性分布,符合哈迪-温伯格定律。     

人谷胱甘肽硫转移酶M1基因在CHO细胞中的表达

目的　构建人谷胱甘肽硫转移酶M1(GSTM1)的CHO细胞表达体系。方法　重组质粒pcDNA3 1 GSTM1用LipofectamineTM2 0 0 0转染CHO细胞 ,用G418筛选细胞的阳性克隆 ,并用PCR、RT PCR和Westernblot进行鉴定 ,测定GSTM1酶活性。结果　GSTM1基因已转染到CHO细胞的基因组 ,并转录表达 ,生成GSTM1蛋白 ,经测定活性达到2 8mmol/ (min·mgprot)。结论　通过CHO GSTM1细胞表达 ,为基因工程生产具有完整功能GSTM1的进一步纯化提供了材料 ,为研究化学致癌物在真核细胞中的代谢 ,作为遗传药理学、毒理学研究的候选细胞株奠定了基础。     

Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant

Our previous studies have shown the adjuvanticity of an Onchocerca volvulus recombinant protein, Ov-ASP-1 (ASP-1), when administered in an aqueous formulation with bystander vaccine antigens or commercial vaccines. In this study, we reported a novel formulation that took advantage of the protein nature of the ASP-1 adjuvant by creating recombinant fusion protein vaccines linking the highly conserved extracellular domain of M2 protein (M2e) consensus sequence of H5N1 influenza viruses with the ASP-1 adjuvant. Two recombinant fusion proteins designated M2e-ASP-1 and M2e3-ASP-1 were studied, in which ASP-1 was fused with one or three tandem copies of the M2e antigen. Our results show that these novel recombinant influenza vaccines, particularly M2e3-ASP-1, induced strong anti-M2e-specific humoral and cellular immune responses in the established mouse model. Furthermore, M2e3-ASP-1 was able to provide significant cross-clade protection against divergent H5N1 viruses. Consequently, this study has demonstrated a potential novel vaccine formulation that could provide a complementary prophylactic strategy in preventing the threat of future influenza outbreak resulting from rapid evolution of the H5N1 virus and co-circulation of multiple antigenic variants in various regions.     

DNA adduct formation of benzo[a]pyrene in white blood cells of workers exposed to polycyclic aromatic hydrocarbons

The major DNA adducts of anti-benzo[a]pyrene diolepoxide (BPDE) were determined by high performance liquid chromatography with fluorescence detection (HPLC-FLD) in white blood cells (WBC) of workers exposed to benzo[a]pyrene (B[a]P). In addition, ambient concentrations of B[a]P at the workplace were determined by personal air sampling. Workers in a refractory setting were examined before (n=26) and 3 months after (n = 33) changing the production material (binding pitch). Furthermore, 9 coke oven workers were examined. The change in the production process in the refractory setting led to a decrease in the median of ambient B[a]P concentrations (0.14 to <0.07 microg/m3). The median of BPDE-DNA adduct levels in WBC also decreased from 0.9 adducts/10(8) nucleotides before changing the production material to <0.5 adducts/10(8) nucleotides 3 months afterwards. The B[a]P concentrations at the workplace for the coke oven workers were found to be significantly higher than in the refractory setting. However, BPDE-DNA adduct concentrations in coke oven workers and refractory setting workers showed no significant difference, which was probably due to the low number of studied subjects in the coke-oven setting. No significant differences could be observed for BPDE-DNA adduct levels between current smokers (n=21) and non-smokers (n=14; p = 0.93) from both plants. In addition, no correlation between B[a]P concentrations in the air and DNA adduct levels in refractory workers and in coke oven workers could be found (r = -0.03, p = 0.87). Because of the missing correlation between personal air sampling and BPDE-DNA adduct levels in WBC, the results may indicate that their formation is either influenced by other routes of exposure to B[a]P (e.g., skin absorption, dietary habits) or interindividual differences in their formation and repair.     

人类免疫缺陷病毒1感染相关的基因多态性在中国汉族人群中的分布

目的　调查中国汉族人群中人类免疫缺陷病毒 1(HIV 1)感染相关的CCR5△ 32、CCR2 6 4I和SDF1 3’A等位基因突变频率和多态性的特点。方法　以 12 5 1例汉族人群为研究对象 ,应用PCR、PCR/RFLP(聚合酶链反应 /限制性片段长度多态性分析 )和DNA直接测序等方法进行检测 ,并用统计学方法进行分析。结果　发现中国汉族人群中存在CCR5△ 32等位基因突变 (均为杂合子基因型 ) ,突变频率为 0 .0 0 119,和西欧及美国白人相比 ,中国人群中CCR5△ 32基因突变频率极低 ,而CCR2 6 4I和SDF1 3’A基因突变频率相对较高 ,分别为 0 .2 0 0 2 3和 0 .2 872 3。结论　中国汉族人群的CCR5△ 32、CCR2 6 4I和SDF1 3’A等位基因的突变和多态性特点 ,具有一定的代表性。由于CCR5△ 32突变率低 ,中国汉族人群对性接触传播的HIV 1病毒 (R5 )株可能有较大的遗传易感性     

人类免疫缺陷病毒1感染相关的基因多态性在中国汉族人群中的分布

目的：调查中国汉族人群中人类免疫缺陷病毒１（ＨＩＶ－１）感染相关的ＣＣＲ５Δ３２、ＣＣＲ２－６４１和ＳＤＦ１－３‘Ａ等位基因突变频率和多态性的特点。方法：以１２５１例汉族人群为研究对象,应用ＰＣＲ、ＰＣＲ／ＲＦＬＰ（聚合酶链反应／限制性片段长度多态性分析）和ＤＮＡ直接测序等方法进行检测,并用统计学方法进行分析。结果：发现中国汉族人群中存在ＣＣＲ５Δ３２等位基因突变（均为杂合子基因型）,突变频率为１     

PPP1R3基因Asp905Tyr多态性与安徽省汉族人群2型糖尿病相关性研究

目的 研究1型蛋白磷酸酶的骨骼肌特异糖原靶向调节亚单位基因(PPP1R3)Asp905Tyr多态性与安徽省汉族人群2型糖尿病相关性。方法 选取安徽省合肥地区汉族2型糖尿病患者262例,健康成人104名,运用聚合酶链反应限制性片段长度多态性技术(PCR-RFLP)进行基因型测定。以体重指数(BMI)25kl/m~2为分割点,将病例组和对照组进行分层分析。结果 PPP1R3基因Asp905Tyr多态性与安徽省汉族人群2型糖尿病没有明显的相关性;以BMI<25kg/m~2基因型Tyr/Tyr组为参照组,BMI≥25kg/m~2携有Asp 905等位基因个体的糖尿病发病风险明显增加(OR=3.69,95％CI:1.38～8.89,P=0.006)。结论 PPP1R3基因Asp 905Tyr多态性可能不是安徽省汉族人群2型糖尿病主要的致病因素。肥胖与Asp905等位基因间的交互作用可增加糖尿病的发病风险。     

Combined effect of MPO, GSTM1 and GSTT1 polymorphisms on chromosome aberrations and lung cancer risk

The role of myeloperoxidase (MPO), and glutathione S-transferase μ and θ (GSTM1 and GSTT1) genetic polymorphisms on lung cancer risk was investigated in 110 Caucasian patients and 119 matched controls. Single genotype variants were not significantly associated with lung cancer risk. However, inheritance of the combined GSTM1 and GSTT1 null genotypes showed a significant increase in risk (crude OR=2.32, 95% CI=1.01 – 6.04). Based on adjustment by age, gender and smoking history, the MPO GA interacted with the presence of GSTM1 and GSTT1 genotypes to significantly reduce the risk (OR=0.17, 95% CI=0.03 – 0.98). From the chromosome aberration (CA) study in a subgroup of 79 patients and 69 matched controls, patients had significantly more CA than the controls. Among the patients, GSTM1 null was associated with a significant increase of CA and MPO AA was associated with a significant decrease of CA compared to their respective wild-type genotypes. After stratifying by smoking history (≤ and>40 pack-years) and genotype, patients still had significantly more CA than the respective controls in most genotype categories. This indicates that the patients had additional contributing factors such as other susceptibility genes and/or different styles of smoking compared with the controls. In conclusion, our study indicates that CA is a useful biomarker to show the functional characteristics of genotypes and the interactive effects from combined genotypes. Therefore, our study strengthens the combined use of genotype and biomarkers for genetic susceptibility to environmental cancer.     

转化生长因子-β1基因多态性与新疆哈萨克族原发性高血压的关系

原发性高血压(EH)是一类由多基因和环境因素共同影响导致的复杂遗传性疾病,是最常见的慢性心血管病之一,其病因和发病机制尚未完全阐明.新疆哈萨克族人群是EH易感人群,居全国各民族高血压患病率的前5位[1].哈萨克族人群居住在气候寒冷,交通不便,相对隔离的山区,无异族通婚史,有着鲜明的民族聚集和高发的特征.目前国内外有关单核苷酸多态性(SNP)与疾病的关联研究,在SNP位点的选择上除了注意功能区(启动子和外显子)外,由于不同连锁标记间连锁不平衡(LD)模式,为增加基因分型的有效性,还需进行标签SNP(tagSNP)与疾病的关联研究.由于转化生长因子-β1(TGF-β1)基因是血压和血管重构的重要调节因子,可能导致高血压的发病,而TGF-β1基因多态性在高血压中的作用尚无确切结果,因此本研究采用SNaPshot技术对TGF-β1基因的tagSNP多态位点开展研究,以期进一步阐明基因型与EH发生的关联.     

江苏省居民电压依赖钾通道蛋白1基因多态性与2型糖尿病的相关性

目的 探讨江苏省居民电压依赖钾通道蛋白1(KCNQ1)基因多态性与2型糖尿病发病的关系.方法 研究对象分别来自于江苏省无锡市2007年2型糖尿病队列研究和南通市2009年慢性非传染性疾病横断面调查,共有病例2925例和对照3281名,对其进行流行病学问卷调查及体格检查,并于清晨采集空腹10 h静脉血5 ml,应用TaqMan OpenArray基因分型系统和i-PLEX SequenomMassARRAY基因分型平台进行KCNQ1基因分型.分析调整年龄、性别和BMI后KCNQ1基因4个位点多态性与2型糖尿病发病风险的关系.结果 调整年龄、性别和BMI后,KCNQ1基因rs2237897、rs2237892、rs2237895位点C等位基因的存在均增加了2型糖尿病的患病风险,相加模型的0R(95％ CI)值分别为1.41(1.30～1.54)、1.35(1.24 ～ 1.47)和1.22(1.12～1.33)(P值均＜0.05).按年龄、性别、BMI进行分层后,各层间rs2237897位点C等位基因增加了2型糖尿病的患病风险,≤56和＞56岁相加模型的0R(95％ CI)值分别为1.39(1.22～1.59)、1.43(1.28～1.60);女性和男性的0R(95％CI)值分别为1.40(1.26～ 1.55)、1.44(1.26～1.66);BMI＜ 24 kg/m2和≥24 kg/m2的OR(95％ CI)值分别为1.48(1.33 ～1.66)、1.34(1.17 ～1.53)(P值均＜0.05).结论 KCNQ1基因rs2237897、rs2237892、rs2237895位点多态性与江苏省汉族居民2型糖尿病的发生相关.     

Cytochrome P4501A induction and DNA adduct formation in glaucous gulls (Larus hyperboreus), fed with environmentally contaminated gull eggs

This study indicates that complex mixtures of pollutants found in the Arctic marine environment have genotoxic effects in glaucous gulls (Larus hyperboreus). DNA adducts were quantified, by the (32)P-postlabeling technique, in liver samples from gulls fed with hen eggs (controls) and from gulls fed with environmentally contaminated gull eggs (exposed). All birds were grown and fed under laboratory conditions. Hepatic homologues to mammalian cytochrome P4501A (CYP1A) proteins were also determined by Western blotting. DNA adducts were detected in all but one liver sample, but the exposed birds had a significantly increased level of DNA adducts relative to that of the controls. There was no clear significant correlation between the DNA adduct level and the level of organochlorine compounds (OCs) in blood. The level of CYP1A protein was significantly higher in the liver of exposed male gulls than in the liver of control males and positively correlated, with significance, to the level of OC compounds measured in blood. There was no significant correlation between the level of DNA adducts and the CYP1A protein content.     

Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent,cross-clade protection against H5N1 influenza virus in mice

H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8⁺ T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity.     

醛固酮合成酶基因单核苷酸多态性与心房颤动的关系

目的 探讨醛固酮合成酶(CVP11B2)基因的单核苷酸多态性与中国汉族人群非家族性心房颤动(房颤)的关系.方法 采用1:1配对的病例对照研究方法,选择297例住院房颤患者作为病例组,同时期、同病房的297例非房颤患者作为对照组.应用GenomeLab~(TM) SNPstream基因分型系统,对CYP11B2基因的两个标签单核苷酸多态位点(tSNPs-rs4545、rs3802228)进行多态性检测.结果 两位点多态性的分布均符合Hardy-Weinberg平衡.病例组患者左心房直径(LA)显著高于对照组(P<0.0001),两个tSNPs的基因型频率及等位基因频率分布在病例组和对照组的差异均无统计学意义,但在病例组3'端非编码区(3'UTR)的rs3802228位点中,具有GG基因型患者的LA显著高于其他型者.多元logistic回归模型在校正年龄、吸烟、BMI、高血压之后,未发现CYP11B2基因两位点基因多态性与房颤发病相关联.两个位点间也不存在连锁不平衡.结论 CYP11B2基因的rs4545tSNPs位点多态性可能与房颤无相关性,位于3'端非编码区的rs3802228tSNPs位点可能与心房结构重构有关.     

Age-related changes in memory and effector T cells responding to influenza A/H3N2 and pandemic A/H1N1 strains in humans

Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. We show that in older compared to young adults, effector memory and effector CD8+ T-cell subsets responding to influenza A/H3N2 challenge have diminished cytolytic activity. In contrast, effector CD4+ T-cell subsets in older adults share similar phenotypic and functional characteristics with those from young adults. Further, we observed a diminished cytolytic T-cell response to both seasonal influenza A/H3N2 and pandemic H1N1 (pH1N1) strains in older compared to young adults who had received seasonal influenza vaccine. These results are consistent with the observed rates of serious complications from seasonal and pandemic influenza infections in different age groups, and suggest that CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process.     

绿茶抑制大鼠体内杂环胺-DNA加合物的形成

食物中的致突变物２－氨基－１－甲基－６－苯基咪唑并「４，５－ｂ」吡啶可诱发大鼠结肠和乳腺肿瘤，而且与人类癌症特别是结肠和直肠癌的密切关系。本研究以致癌物－ＤＮＡ加合物为生物标记物，观察绿茶对ＰｈＩＰ致癌作用的预防效果及机理。     

亚洲人群中SDF1-3’A和CCR2-64I基因的多态性与HIV-1感染相关性的Meta分析

目的探讨SDF1-3’A和CCR2-64I基因的多态性与HIV-1感染之间的关联性。方法检索国内外有关SDF1-3’A和CCR2-64I基因的多态性与HIV感染相关性的病例对照研究文献进行Meta分析。结果符合CCR2-64I入选条件的文献有13篇,20个研究,总共有5578例样本,其中病例有2898例,健康对照有2680例,当CCR2-64I基因所入选的研究全部合并在一起时,在所有模型中没有发现其显著相关性（CY vs. CC: OR = 0.97,95% CI = 0.85-1.09; CY + YY vs. YY: OR = 1.00,95% CI = 0.89-1.13;YY vs. CC：OR = 1.28,95% CI = 0.97-1.69）。符合SDF1-3’A入选条件的文献13篇,19个研究,总共有4854例,其中病例有2281例,健康对照有2573例,在整个和亚组分析中没有发现其显著相关性。而在印度人群发现高度的异质性。结论亚洲人群CCR2-64I两种抗性基因与HIV-1感染之间无相关性;中国人群SDF1-3’A与HIV-1感染之间无相关性;印度人群SDF1-3’A与HIV-1感染之间是否有相关性仍需作进一步的研究。