【作者】 刘琳琳；

【导师】 吴志刚；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2008， 硕士

【摘要】 苯并(a)芘[benzo(a)pyrene, B(a)P],又名3,4-苯并芘,由一个苯环和一个芘分子结合而成,是一种广泛存在于人们生产、生活环境中的污染物。1933年,英国学者从煤焦油中分离出苯并芘(B(a)P),并成功诱发出小鼠皮肤癌,使B(a)P成为第一个被发现的环境化学致癌物。动物实验证明,B(a)P对局部或全身都有致癌作用;流行病学研究表明,B(a)P可以通过皮肤、呼吸道、消化道等多种途径被人体吸收,能够诱发皮肤癌,肺癌,直肠癌,胃癌,膀胱癌等。除致癌外,B(a)P还具有致畸,致突变作用,对人体内分泌系统也有一定的干扰作用。1983年国际癌症机构将其确认为环境致癌物。B(a)P虽然被认为是高活性致癌剂,但并非直接致癌物,必须经细胞微粒体中的混合功能氧化酶激活才具有致癌性。B(a)P在体内首先经混合功能氧化酶细胞色素P450酶(CYP1A1)催化在结构弯区形成7,8环氧苯并芘,然后在环氧化物酶和CYP1B1作用下形成7,8二氢二醇苯并芘,该物质经混合功能氧化酶催化,形成二氢二醇环氧苯并(a)芘(BPDE)。BPDE能够与与体内生物大分子如DNA,蛋白质等结合生成共价化合物,被认为是B(a)P在体内的终致癌物。因此随着B(a)P越来越深入的研究,BPDE的致癌性也受到广泛关注,但是具体作用机制尚不明确。BPDE主要与DNA的小沟上的亲核位点鸟嘌呤的环外胺基端共价结合,产生特异突变,形成BPDE-N2-dG。BPDE加合物能够阻止DNA复制,造成复制阻断,复制准确性降低,诱发加合物对应位点上的碱基误渗,最终出现G-T的转变及部分G-A的错误突变,这既是癌变的启动因子,又在癌变发展过程起着关键的作用。B(a)P主要存在于煤焦油、各类碳,石油等燃烧时产生的烟气、汽车尾气中,还有工业废水中及吸烟时香烟产生的烟雾中。另外烧烤和熏制食品也含有大量的B(a)P。因此除了职业暴露外,我们在生活中也会经常接触到B(a)P。鉴于B(a)P对人类的危害性,这几十年来已经有大量的研究报道了B(a)P的终致癌物质BPDE的检测方法,但其绝大部分的研究都是着重于检测BPDE的DNA﹑蛋白加合物或BPDE加合物的水解产物进行定性,定量分析,这些方法前处理步骤繁多致使BPDE加合物的回收率低。因此如果能够选择直接BPDE,不仅简单易行,可以提供个体“内接触”量或“有效接触”量,还能提供化学毒物的代谢活化信息。在本研究中,采用高效液相色谱-紫外检测和荧光检测法对BPDE标准品溶液及细胞液中的BPDE进行检测,为进一步研究BPDE的定性和定量分析奠定基础;用单细胞凝胶电泳方法检测了BPDE对体外培养的HepG2细胞的DNA损伤,以期反映BPDE对遗传物质的损伤;最后是对B(a)P的原型消减进行探索性研究,用B(a)P单独染毒或B(a)P与PCB153联合染毒HepG2细胞,然后用HPLC-UVD﹑FD检测经HepG2细胞代谢后细胞中的B(a)P的浓度,探索PCB153在B(a)P代谢过程中的作用。第一部分B(a)P代谢产物BPDE的检测技术1. B(a)P代谢产物BPDE的HPLC-紫外检测技术采用高效液相色谱-紫外测定的方法检测BPDE标准品溶液及在细胞液中的BPDE。结果发现:BPDE标准品在DMSO溶液中的浓度与其峰面积呈现出良好的线性关系,其线性回归方程为Y=637138X-124587,R2=0.9982。BPDE标准品在浓度1.00～20.00μg/ml范围内具有良好的线性关系,该方法的最低检出限为0.0625μg/ml。BPDE标准品在细胞裂解液中的线性回归方程为Y=424298X+24598,R2=0.9921。本方法的日内精密度RSD 2.9～3.6%和日间精密度RSD 4.3～5.9%。BPDE化学性质极不稳定,对光,湿度及酸很敏感。在同样的浓度下,BPDE标准品和细胞裂解液中的BPDE标准品的峰面积出现差异,经过分析,在细胞裂解液中的BPDE的含量偏低,但显著高于在环境中的自然变化。2. B(a)P代谢产物BPDE的HPLC-荧光检测技术采用高效液相色谱-荧光测定方法检测BPDE标准品溶液及在细胞液中的BPDE。结果发现:BPDE标准品在DMSO溶液中的浓度与其峰面积呈现出良好的线性关系,其线性回归方程为Y=2E+07X+178569,R2=0.9988。BPDE标准品在浓度0.01～1.00μg/ml范围时具有良好的线性关系,该方法的最低检出限为0.005μg/ml。本方法的日内精密度RSD 3.2～4.6%和日间精密度RSD 5.9～7.4%。在同样的浓度下,BPDE标准品和细胞裂解液中的BPDE标准品的峰面积出现差异,经过分析,在细胞裂解液中的BPDE的含量偏低,但显著高于在环境中的自然变化。第二部分BPDE致HepG2细胞DNA损伤的毒理学研究用0.05μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,2μmol/L5个不同浓度的BPDE染毒体外培养的HepG2细胞,用50μmol/LB(a)P做阳性对照,采用单细胞凝胶电泳的方法检测BPDE对HepG2细胞的DNA损伤。结果显示,在本实验中不同浓度的BPDE染毒组与正常组相比Olive尾矩均有显著性差异(P<0.01),而不同浓度组之间差异也有显著性(P<0.01),BPDE最高浓度组与B(a)P阳性对照组相比也有显著性差异(P<0.01),随着BPDE浓度的增加,HepG2细胞的Olive尾矩值逐渐增大,并呈明显的剂量-依赖关系。这说明反式BPDE可引起DNA断裂损伤,且损伤程度与染毒剂量有关。第三部分苯并(a)芘原型代谢消减的探索性研究用50μmol/L B(a)P对HepG2细胞单独作用72个小时或先用不同浓度(0.1μmol/L, 1μmol/L,10μmol/L,100μmol/L)PCB153对HepG2细胞预处理48个小时然后再与50μmol/L B(a)P联合作用24个小时,采用HPLC-UVD﹑FD检测HepG2细胞内经过代谢后的B(a)P的浓度。结果显示,PCB153在低浓度0.1μmol/L和1μmol/L与B(a)P联合作用时,在HepG2细胞内检测到的B(a)P浓度要高于B(a)P单独作用的浓度,且随着PCB153浓度的增高,在HepG2细胞内检测到的B(a)P浓度有增高的趋势。但是在PCB153的浓度为10μmol/L和100μmol/L时,它与B(a)P的联合作用后在HepG2细胞内检测到的B(a)P浓度要低于B(a)P单独作用的浓度,且随着PCB153浓度的增高,在HepG2细胞内检测到的B(a)P浓度呈现出降低的趋势。由此我们推测PCB153在诱导P450酶活性时可能存在一定的浓度范围,在浓度10μmol/L～100μmol/L PCB153可以增强P450酶活性,从而可以进一步促进B(a)P在细胞内的代谢,低于此范围则可能不会增强P450酶活性。更多还原

【Abstract】 B(a)P was also named 3,4- benzo(a)pyrene, composed of a benzene ring and a pyrene molecule, and was a pervasive chemical pollutant in our industrial and living environment. In 1933, the British scholar dissociated benzo(a)pyrene from coal tar, and succeeded in inducing the skin cancer of mice, making benzo(a)pyrene become the first discovered environmental chemical carcinogen. It was identified from generous animal experiments that B(a)P exhibits carcinogenic properties in the part or all of the body. Epidemiologic studys signed that there were many ways that B(a)P could be absorpted, such as passing skin, respiratory passage and so on. Besides carcinogenicity,B(a)P also could cause aberration and mutagenesis. Moreover, it was thought to interfere with endocrine system of human. In 1983, the International Agency for Research on Cancer confirmed B(a)P to be an environmental carcinogen.Although B(a)P was thought to be carcinogenic agent with high activity, it was not the direct acting carcinogen, and it must be activated by mixed function oxidase in the microsome to possess the ability of carcinogenesis. B(a)P was metabolically activated via a three-step process. First, cytochromes P450 catalyze the formation of (7R,8S)-epoxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-oxide). This is converted to (7R,8R)-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-diol), catalyzed by epoxide hydrolase. BaP-7,8-diol then undergoes another oxidation step, catalyzed by cytochromes P450 and other enzymes, producing mainly (7R,8S)-dihydroxy-(9S,10R)-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). BPDE reacted with biomacromolecule in the body, such as DNA, protein, producing covalent compound, so BPDE was believed to be the ultimate genotoxic metabolite of B(a)P. With the development of studies in B(a)P, popeple payed close attention to the carcinogenicity of BPDE, but the specific mechanism of action was still indefinite.BPDE reacted with DNA producing a major adduct at the N2 position of deoxyguanosine (BPDE-N2-dG). BPDE adducts could prevent DNA from duplicating, result in blocking the duplication, decrease accuracy of duplication and induce the incorrect leak of bases in corresponding site which cotained adducts, at last there would be appeared G-T transformation and partial G-A mutation. So BPDE adducts were thought to play an important role in the early stages of carcinogenesis, and also in the process of growth of cancer.B(a)P was commonly found in coal tar, the smoke from burning carbon, coal and petroleum, vehicle exhaust fumes, industrial waste and tobacco smoke. In addition, broiled and smoked foods also contained considerable B(a)P. So except occupational exposure, in the living enviroment we usually got touch with B(a)P. In light of healthy perniciousness of human from BPDE, which was the ultimate genotoxic metabolite of B(a)P, a plethora of studies had addressed the detection of DNA and protein adducts in human, cell lines and animal exposed to BPDE, but many of them layed particular emphasis on qualitative and quantitative analysis of BPDE-DNA adducts, protein adducts or hydrolysis products of adducts, which the pretreatment were too many so that the recovery rates of BPDE adducts were prone to low. If we choosed directly detect BPDE, not only the methods were simple, easily to operate and could be served as molecular biomarkers suitable for use in risk assessment, but also provided the metabolism activated information of chemical poisons.In this study, we detected the purified BPDE in DMSO solution and BPDE in cell lysate using high performance liquid chromatography with ultraviolet detector (HPLC-UVD) and high performance liquid chromatography with fluorescence detector (HPLC-FD), settling a good foundation to investigate qualitation and quantitative analysis of BPDE further; then detected BPDE induced the DNA damage of HepG2 cell cultured in vitro by single cell gel electrophoresis, which were reflected the damage of genetic material from BPDE; at last explored metabolism of B(a)P prototype, HepG2 cells were treated with B(a)P alone, or pretreated with different concentrations of PCB153 then co-treated with B(a)P and PCB153, then using high performance liquid chromatography with ultraviolet detector and fluorescence detector, detected the density of B(a)P metabolismed in HepG2 cells, with the hope of illuminating the effect of PCB153 in the metabolic process of B(a)P.PartⅠTechniques of detecting BPDE metabolized from B(a)P1. Technique of detecting BPDE metabolized from B(a)P with HPLC-UV detectorWe detected purified BPDE in DMSO solution and BPDE in cell lysate using high performance liquid chromatography with ultraviolet detector (HPLC-UVD). The results were as follows: it was showed a good linear correlation between the concentrations of BPDE in DMSO solution and its peak areas, the linear response with the concentration of BPDE in DMSO solution and its peak area was obtained at the range of 1.00~20.00μg/ml, the equation of linear regression was Y=637138X-124587, the correlation coefficient was 0.9982, the lowest detection limit was 0.0625μg/ml. The equation of linear regression of BPDE in cell lysate was Y=424298X+24598, the correlation coefficient was 0.9921. The intra-assay and inter-assay relative standard deviations were 2.9～3.6% and 4.3～5.9%, respectively. BPDE chemical property was extremely instable and was sensitive to light, moisture and acidic PH. In the same density, the areas between purified BPDE and BPDE in cell lysate were different. According to the data, we discovered the content of BPDE in in cell lysate was lower than that of purified BPDE, but significantly higher than the natural change in the environment.2. Technique of detecting BPDE metabolized from B(a)P with HPLC-fluorescence detectorWe detected purified BPDE in DMSO solution and BPDE in cell lysate using high performance liquid chromatography with fluorescence detector (HPLC-FD). The results were as follows: it was showed a good linear correlation between the concentrations of BPDE in DMSO solution and its peak areas, the linear response with the concentration of BPDE in DMSO solution and its peak area was obtained at the range of 0.01～1.00μg/ml, the equation of linear regression was Y=2E+07X+178569, the correlation coefficient was 0.9988, the lowest detection limit was 0.005μg/ml. The equation of linear regression of BPDE in cell lysate was Y=424298X+24598, the correlation coefficient was 0.9921. The intra-assay and inter-assay relative standard deviations were 3.2～4.6% and 5.9～7.4%, respectively. In the same density, the areas between purified BPDE and BPDE in cell lysate were different. According to the data, we discovered the content of BPDE in in cell lysate was lower than that of purified BPDE, but significantly higher than the natural change in the environment.PartⅡToxicological study on BPDE induced DNA damage in HepG2 cell HepG2 cells were treated with different concentrations of BPDE (0.05μmol/L, 0.1μmol/L, 0.5μmol/L, 1μmol/L, and 2μmol/L) for 24 hours. 50μmol/L B(a)P was used as positive control. The damage of HepG2 cells were detected by single cell gel electrophoresis. The results showed that: in this part of study, the significant differences of Olive tail moment were found between the different concentrations groups of BPDE and normal group(P<0.01), and the significant differences of Olive tail moment were also among the groups of the different concentrations BPDE(P<0.01), in the same time, the significant difference was present between the highest BPDE and positive control. BPDE caused increase in Olive tail moment in a dose-dependent fashion. Statistical significant increases of Olive tail moment were observed in HepG2 cell treated with 0.05, 0.1, 0.5, 1 and 2μmol/L BPDE for 24 hours. These results demonstrated that BPDE could induce DNA damage which were in a dose-dependent fashion.PartⅢThe exploratory study on the metabolism of B(a)P prototypeHepG2 cells were treated with 50μmol/L B(a)P for 72 hours alone, or pretreated with different concentrations of PCB153(0.1μmol/L, 1μmol/L, 10μmol/L, 100μmol/L) for 48 hours then co-treated with 50μmol/L B(a)P and PCB153 for another 24 hours. The density of B(a)P metabolismed in the HepG2 cells were detected by high performance liquid chromatography with ultraviolet detector and fluorescence detector (HPLC-UVD﹑FD).The results were as follows: Compared with group treated with B(a)P alone, enhanced concentrations of B(a)P detected in the HepG2 cells were found in combined treatment groups which PCB153 were at low concentrations (0.1μmol/L, 1μmol/L), and with increasing the concentrations of PCB153, the concentrations of B(a)P detected in the HepG2 cells were also increasing. Although at high concentrations of PCB153 (10μmol/L, 100μmol/L), the concentrations of B(a)P detected in the HepG2 cells were lower than that in the group treated with B(a)P alone , which were decreased with increasing the concentrations of PCB153. So we supposed that when PCB153 induced the enzyme activity of P450, there would be some range of density. Between the range of 10μmol/L and 100μmol/L, the enzyme activity of P450 could be enhanced by PCB153, thus enhanced the metabolism of B(a)P in the HepG2 cells. Once out of this range, PCB153 could not enhance the enzyme activity of P450.更多还原