Beland Frederick A, Churchwell Mona I, Von Tungeln Linda S, Chen Shoujun, Fu Peter P, Culp Sandra J, Schoket Bernadette, Gyorffy Erika, Minárovits János, Poirier Miriam C, Bowman Elise D, Weston Ainsley, Doerge Daniel R
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Chem Res Toxicol. 2005 Aug;18(8):1306-15. doi: 10.1021/tx050068y.
A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.
开发并验证了一种使用高效液相色谱（HPLC）结合电喷雾串联质谱（ES-MS/MS）来检测和定量主要DNA加合物的方法，该加合物由暴露于最终致癌性苯并[a]芘（BP）代谢物反式-7,8-二羟基-反式-9,10-环氧-7,8,9,10-四氢苯并[a]芘（BPDE）产生。将小牛胸腺DNA与BPDE反应，酶解为核苷，主要的DNA加合物10-(脱氧鸟苷-N2-基)-7,8,9-三羟基-7,8,9,10-四氢苯并[a]芘（dG-BPDE）通过HPLC纯化。采用类似程序从[1,2,3,4,5,6,11,12-(2)H8]BPDE制备dG-BPDE-d8用作内标。使用水解的鲑鱼精巢DNA（82微克）和10皮克dG-BPDE（相当于6.9个加合物/10(8)个核苷酸）的混合物对HPLC-ES-MS/MS方法进行验证。结果表明日间和日内准确度为99 - 100%，精密度为1.6 - 1.7%（相对标准偏差）。当应用于用[1,3-(3)H]BPDE体外修饰的小牛胸腺DNA样品时，该方法得到的值与通过放射性标记、32P后标记和免疫测定获得的值非常相似。对腹腔注射0.5和1.0毫克[7,8-(3)H]BP的小鼠肝脏DNA进行HPLC-ES-MS/MS分析，得到的值与通过32P后标记和免疫测定确定的值相当。喂食含0.3%煤焦油饮食（约含2毫克BP/克煤焦油）一个月的小鼠肺DNA有0.6±0.04个dG-BPDE加合物/10(8)个核苷酸。该值远低于通过32P后标记测定的102±14个总DNA加合物/10(8)个核苷酸，这表明dG-BPDE对给予煤焦油的小鼠肺组织中形成的DNA加合物贡献很小。使用HPLC-ES-MS/MS方法评估人肺DNA样品中dG-BPDE的存在情况。基于检测限为0.3个dG-BPDE加合物/10(8)个核苷酸，当使用100微克DNA时，26个样品中仅在1个样品中检测到dG-BPDE。这些观察结果表明HPLC-ES-MS/MS适用于评估BP对多环芳烃（PAH）混合物暴露所致DNA损伤的贡献。结果进一步表明，在暴露于PAHs的个体中，dG-BPDE可能仅占其他DNA加合物检测方法所检测到的总DNA加合物的一小部分。
