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.
Bibliographical noteFunding Information:
*E-mail: email@example.com. Phone: 612-626-0141. ORCID Robert J. Turesky: 0000-0001-7355-9903 Funding This research was supported by Grant No. R01ES019564 (R.J.T.) from the National Institute of Environmental Health Sciences, R01CA122320 (R.J.T.), and R01 CA220367 (R.J.T. and T.A.R.), and R33CA186795 (R.J.T.) from the National Cancer Institute of the National Institutes of Health, and from Henry and Marsha Laufer (A.P.G., K.G.D., and T.A.R.). Mass spectrometry was carried out in Analytical Biochemistry Shared Resources of the Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant No. CA-077598. Notes The authors declare no competing financial interest.
We thank Dr. Frederick A. Beland from the National Center for Toxicology Research/US FDA for providing PhIP and 4-ABP treated CT-DNA. We thank Dr. Badrinath Konety, MD, Department of Urology, University of Minnesota, for the interest and support of this project. Dr. Suprita Krishna and Resha Tejpaul, from the Department of Urology, are acknowledged for administrative assistance. Drew Sciacca, Department of Laboratory Medicine and Pathology, who handled the prostatectomy specimens and Beth Fenske and Carla Heinke, from BioNet Tissue Procurement, for the collection of the prostate biospecimens are thanked. The deidentified renal cortex specimens were kindly provided by Dr. Bojan Jelakovic,́ School of Medicine, University of Zagreb, Croatia. Technical support was provided by the Stony Brook University Research Histology Core and is gratefully acknowledged.