Yun Byeong Hwa, Xiao Shun, Yao Lihua, Krishnamachari Sesha, Rosenquist Thomas A, Dickman Kathleen G, Grollman Arthur P, Murugan Paari, Weight Christopher J, Turesky Robert J
Masonic Cancer Center, Division of Carcinogenesis and Chemoprevention and Department of Medicinal Chemistry, ‡Department of Laboratory Medicine and Pathology, and §Department of Urology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Department of Pharmacological Sciences and ¶Department of Medicine, Stony Brook University , Stony Brook, New York 11794, United States.
Chem Res Toxicol. 2017 Dec 18;30(12):2130-2139. doi: 10.1021/acs.chemrestox.7b00218. Epub 2017 Nov 27.
Formalin-fixed paraffin-embedded (FFPE) tissues are rarely used for screening DNA adducts of carcinogens because the harsh conditions required to reverse the formaldehyde-mediated DNA cross-links can destroy DNA adducts. We recently adapted a commercial silica-based column kit used in genomics to manually isolate DNA under mild conditions from FFPE tissues of rodents and humans and successfully measured DNA adducts of several carcinogens including aristolochic acid I (AA-I), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Yun et al. (2013) Anal. Chem. 85, 4251-8, and Guo et al. (2016) Anal. Chem. 88, 4780-7). The DNA retrieval methodology is robust; however, the procedure is time-consuming and labor intensive, and not amenable to rapid throughput processing. In this study, we have employed the Promega Maxwell 16 MDx system, which is commonly used in large scale genomics studies, for the rapid throughput extraction of DNA. This system streamlines the DNA isolation procedure and increases the sample processing rate by about 8-fold over the manual method (32 samples versus 4 samples processed per hour). High purity DNA is obtained in satisfactory yield for the measurements of DNA adducts by ultra performance liquid chromatography-electrospray-ionization-ion trap-multistage scan mass spectrometry. The measurements show that the levels of DNA adducts of AA-I, 4-ABP, and PhIP in FFPE rodent and human tissues are comparable to those levels measured in DNA from matching tissues isolated by the commercial silica-based column kits and in DNA from fresh frozen tissues isolated by the conventional phenol-chloroform extraction method. The isolation of DNA from tissues is one major bottleneck in the analysis of DNA adducts. This rapid throughput methodology greatly decreases the time required to process DNA and can be employed in large-scale epidemiology studies designed to assess the role of chemical exposures and DNA adducts in cancer risk.
福尔马林固定石蜡包埋（FFPE）组织很少用于筛查致癌物的DNA加合物，因为逆转甲醛介导的DNA交联所需的苛刻条件会破坏DNA加合物。我们最近采用了一种基因组学中常用的基于硅胶柱的商业试剂盒，在温和条件下从啮齿动物和人类的FFPE组织中手动分离DNA，并成功测量了几种致癌物的DNA加合物，包括马兜铃酸I（AA-I）、4-氨基联苯（4-ABP）和2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶（PhIP）（Yun等人，（2013年）《分析化学》85卷，4251 - 4258页；以及Guo等人，（2016年）《分析化学》88卷，4780 - 4787页）。DNA提取方法很可靠；然而，该过程耗时且劳动强度大，不适合高通量处理。在本研究中，我们采用了Promega Maxwell 16 MDx系统，该系统常用于大规模基因组学研究，用于高通量提取DNA。该系统简化了DNA分离程序，与手动方法相比，样品处理速率提高了约8倍（每小时处理32个样品对4个样品）。通过超高效液相色谱 - 电喷雾电离 - 离子阱 - 多级扫描质谱法测量DNA加合物时，可获得高纯度DNA，产量令人满意。测量结果表明，FFPE啮齿动物和人类组织中AA-I、4-ABP和PhIP的DNA加合物水平与通过基于硅胶柱的商业试剂盒从匹配组织中分离的DNA以及通过传统酚 - 氯仿提取法从新鲜冷冻组织中分离的DNA中测得的水平相当。从组织中分离DNA是DNA加合物分析中的一个主要瓶颈。这种高通量方法大大减少了处理DNA所需的时间，可用于大规模流行病学研究，以评估化学暴露和DNA加合物在癌症风险中的作用。
