【作者】 张忠彬；

【导师】 夏昭林； 金锡鹏；

【作者基本信息】 复旦大学 ， 卫生毒理学， 2005， 博士

【摘要】 苯广泛应用于医药、化工、喷漆和制鞋等行业,其慢性毒性主要表现为骨髓造血功能的抑制,出现白细胞减少、再生障碍性贫血甚至白血病。在毒物代谢酶的作用下,进入体内的苯代谢活化形成与苯毒性密切相关的环氧化物、酚类和醌类化合物。通过对DNA的直接氧化作用,并与之形成加合物,它们可造成DNA的损伤。DNA损伤后,细胞主要通过碱基切除修复、核酸切除修复、重组修复和错配修复等途径来修复DNA损伤,这一过程涉及到参与修复DNA氧化损伤的基因(hOGG1、hMYH、hMTH1)、切除苯代谢物与体内大分子形成的加合物的基因(APE1、XPD)、修复DNA单链断裂损伤的基因(APE1、XRCC1、ADPRT)和修复DNA双链断裂损伤的基因(XRCC2、XRCC3)等。在这些基因内部存在有大量的单核苷酸多态位点,这些多态可以引起编码产物空间构象或者酶活性的改变,也可以引起编码产物表达量的改变,从而影响到机体修复DNA损伤的能力,与个体慢性苯中毒的发病风险密切相关。 为探讨DNA损伤修复基因多态性、苯接触水平和生活方式与慢性苯中毒遗传易感性的关系,我们设计了本次病例一对照研究。病例组为来自上海、广州、杭州和马鞍山的152名苯中毒工人,152名调查时正在从事接苯作业而没有慢性苯中毒表现的工人为对照组。采用Dosmeci等的评估方法,我们对研究对象的累积接触水平进行了估测,并调查了他们吸烟、饮酒等情况。职业流行病学调查结果表明,病例组和对照组的一般特征如性别、民族、年龄、工龄和累积接触评分在两组人群中的分布无统计学差异,表明病例组和对照组均衡可比。 为了对不能与前后序列构成酶切位点的单核苷酸多态性(SNP)进行有效检测,我们研究了创造酶切位点的限制性片断长度多态检测技术(CRS-RFLP)检测单核苷酸多态性的方法及其应用价值。结果表明CRS-RFLP是检测基因SNP的一种简便易行的有效方法,拓宽了RFLP的应用范围并很好地保持了该方法的一些优点。 应用聚合酶链反应-限制性片断长度多态检测技术(PCR-RFLP)和CRS-RFLP技术,我们研究了上述可能与慢性苯中毒发生关系密切的DNA损伤修复基因的常见多态与个体慢性苯中毒遗传易感性的关系,同时探讨了苯接触强度、生活方式等对个体慢性苯中毒发病风险的影响。结果表明: (1) 未检出XRCC2 Arg188His、XPD Met199Ile和XPD Tyr201His多态的突变基因型,XRCC3 Thr241Met、XPD Asp312Asn、hMTH1 Va183Met、hOGG1 Ser326Cys等多态的突变基因型的频率与国外研究结果存在差异,可能是不同种更多还原

【Abstract】 Benzene is commonly used to synthesize organic chemicals and used as an important component of many organic solvents. The major industries using benzene are those involving in the production of rubber, paint, shoes, lubricants, dyes, detergents, drugs, and pesticides. Long term exposure to benzene could result in chronic benzene poisoning (CBP) characterized by hematotoxicities, mainly pancytopenia, aplastic anemia, myelodysplastic syndrome, and acute myeloid leukemia. Catalyzed by toxicant-metabolizing enzymes, inhaled and absorbed benzene is converted to benzene oxide, which is ultimately metabolized to phenol, catechol, hydroquinone and benzoquinone. Previous studies have demonstrated that benzene toxicity mainly resulted from its intermediate reactive metabolites which lead to DNA damage by directly oxidative damage and covalently binding to DNA. Then the repair systems will complete repair of damaged DNA by means of base excision repair (BER), nucleotide excision repair (NER), double strands break (DSB) repair and mismatched repair (MMR). During period of DNA repair, a great many of DNA repair genes related to repair oxidative damage, adduct damage, single-strand break damage and double-strand break of DNA are involved in. A number of single nucleotide polymorphisms (SNPs) have been identified in these genes, some of which are relatively common and can result in change of enzyme activity or the configuration, the quantity of expressed mRNA as well, which will affect their capability of repairing damaged DNA and the risk of CBP.In order to elucidate their roles in human susceptibility to CBP and effects of benzene exposure duration and life styles, a case-control study was designed and conducted. 152 CBP patients come from Shanghai, Guangzhou, Hangzhou and Maanshan and 152 workers without poisoning manifestations but occupationally exposed to benzene were investigated. The cumulative exposure level was estimated with method described by Dosmeci, and the lifestyles such as cigarette smoking and alcohol consumption were also explored. The results of occupational epidemiology showedthat there was no statistic difference for distribution of sex, race, age, workage and cumulative exposure level in case and control groups, which indicated that it was comprehensive equilibrium for the case and control groups.To determine these SNPs that could not compose sites recognized by restriction enzymes with adjacent sequences effectively, we investigated technique of introducing mismatched bases to create restriction site combined with restriction fragment length polymorphism (CRS-RFLP)and its application. The results suggested that this method is good for identifying found single nucleotide polymorphisms extensively existing in sequence of genes.With method of polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP) and CRS-RFLP, SNPs in XRCC1, XRCC2, XRCC3, ADPRT, APE1, XPD, hMTH1, hOGG1 and hMYH were genotyped. And the associations of these genetic polymorphisms with risk of developing CBP and the modifying effects of intensity of benzene exposure and life styles were also investigated in this case-control study. The results indicated that:(1) No variant allele was detected for polymorphisms of XRCC2 Arg188His、 XPD Met199Ile and XPD Tyr201His. The frequencies of the variant allele for polymorphisms of XRCC3 Thr241Met, XPD Asp312Asn, hMTHl Val83Met and hOGGl Ser326Cys were different from these reported in studies with foreign population, and the frequencies of the variant allele for polymorphisms of XPD Asp312Asn, hOGGl Ser326Cys and ADPRT Val762Ala were not consisted with these reported in other domestic researches.(2) The proportion of XRCC1c. 194Arg/Trp+Trp/Trp genotypes in the case group was lower than that of the control group (50.69% vs 62.22%), and there was a 0.60-fold reduced risk of CBP for individuals carrying XRCC1c.194Arg/Trp+Trp/Trp genotypes (ORadj=0.60, 95%CI: 0.37-0.98, P=0.04) compared with the subjects carrying Arg/Arg allele. The proportion of XPDc. 312Asp/Asn+Asn/Asn genotypes in the case group was also lower than that of the control group (26.90% vs 36.50%). Compared with the subjects carrying XPDc.312Asp/Asp allele, the risk of CBP for the individuals carrying XP Dc.312Asp/Asn+Asn/Asn also decreased (ORad=0.59, 95%CI: 0.35-0.99, P<0.05).(3)The proportion of XRCC1c.280Arg/His+His/His, hMTH1c.83Val/Met+Met/Met and hOGG1c.326 Cys/Cys genotypes in the case group was higher than that of thecontrol group (52.08% vs 36.03%, 16.78% vs 7.30% and 62.68% vs 44.91%, respectively). There was a 1.91-fold increased risk of CBP for the individuals carrying XRCC1c.280 Arg/His+His/His genotypes compared with the subjects carrying Arg/Arg allele (ORadj=1.91,95%C7: 1.17-3.10, p=0.01). Compared with the subjects carrying hMTH1c.83Val/Val allele, there was a 2.51-fold increased risk of CBP for individuals carrying hMTHlc.83Val/Met+Met/Met genotypes (ORadj=2.51, 95%C7: 1.14-5.49, P=0.02). Compared with the subjects carrying hOGG1c.326 Ser/Cys+ Ser/Ser genotypes, there was a 2.49-fold increased risk of CBP for the individual carrying hOGG1c. 326 Cys/Cys allele (ORadj=2.49, 95%CI: 1.52-4.07, P<0.01).(4) Potential interactions were found among smoking and polymorphism of hMYH His335Gln (xh2=4.59, p=0.03), alcohol consumption and polymorphism of APE1 Aspl48Glu (xh2=6.93, P=0.01), intensity of benzene exposure and polymorphism of XPD Asp312Asn (xh2=8.45, p=0.02). Compared with individuals with hMYHc.335 His/His genotype together with habit of smoking, there was a 0.20-fold decreased risk of CBP for the subjects carrying genotypes of hMYHc.335His/Gln+Gln/Gln and smoking at the same time (OR=0.20, 95%CI: 0.05-0.77, p=0.02). There was a 4.13-fold increased risk of CBP for the drinker with genotype of APE1c. 148Asp/Asp compared with these with genotype of APE1c.148AsplAsp without habit of alcohol consumption(OR=4.13, 95%CI: 107-15.85, p=0.03). However, due to the relative few smokers and alcohol users in the subjects, further study needed to elucidate interactions among lifestyles and different polymorphisms. In the low intensity of benzene exposure group, compared with these with XPD c.3\2Asp/Asp, the risk of CBP for these carrying XPDc.312Asp/Asn+Asn/Asn genotypes further decrease (OR=0.19, 95%CI: 0.07-0.53, p<0.01).(5) Stratified analysis of different genetic polymorphisms suggested that there are potential interactions among different genetic polymorphisms (XRCCl Arg280His and XPD Asp312Asn, XRCCl Arg399Gln and hMYH His335Gln, ADPRT Val762Ala and hMYH His335Gln, XPD Asp312Asn and hOGG1 Ser326Cys, hOGG1 Ser326Cys and hMYH His335Gln, respectively). The risk of CBP may decrease for the individuals carrying genotypes of XRCC1c.399Arg/Arg and hMYHc.335His/Gln+Gln/Gln (OR=0.36, 95%CI:0.16-0.83, p=0.02), XRCC1c.399Arg/Gln+Gln/Gln and hMYHc.335 His/His (OR=0.39, 95%CI: 0.16-0.91, p=0.03), hOGG1c.326Ser/Ser+ Ser/Cys and hMYHc.335His/Gln+Gln/Gln (OR = 0.38,95%C/.0.18-0.79, P<0.01) at the same time, respectively. Alternatively, the risk of CBP may further increase for the subjects with更多还原